Methods and Compositions for Treating Neuroblastoma

ABSTRACT

Methods and compositions for treating neuroblastoma are disclosed.

This application is a continuation-in-part of 12/853,834, filed on Aug.10, 2010, which is a continuation-in-part of PCT/US2009/034288, filed onFeb. 17, 2009, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/029,212, filed on Feb. 15, 2008and to U.S. Provisional Patent Application No. 61/123,775, filed on Apr.11, 2008. The foregoing applications are incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates to the fields of neuroblastoma. Morespecifically, the invention provides compositions and methods for theidentification, diagnosis, and treatment of neuroblastoma.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains.

Each of these citations is incorporated herein by reference as thoughset forth in full.

Neuroblastoma is a cancer of early childhood that arises from thedeveloping autonomic nervous system. It is the most common malignancydiagnosed in the first year of life and shows a wide range of clinicalphenotypes with some patients having tumors that regress spontaneously,whereas the majority of patients have aggressive metastatic disease(Maris et al. (2007) Lancet 369:2106-20). These latter neuroblastomacases have survival probabilities of less then 40% despite intensivechemoradiotherapy, and the disease continues to account for 15% ofchildhood cancer mortality (Maris et al. (2007) Lancet, 369:2106-20;Matthay et al. (1999) N. Eng. J. Med., 341:1165-73). Tumors frompatients with an aggressive phenotype often show amplification of theMYCN oncogene (Schwab et al. (1984) Nature, 308:288-91), and/ordeletions of chromosome arms 1p and 11q (Attiyeh et al. (2005) N. Engl.J. Med., 353:2243-53). However, because MYCN is so aberrantlydysregulated, and no putative tumor suppressor gene at 1p and 11 q hasbeen shown to harbor inactivating mutations in more than a smallpercentage of cases, no tractable molecular target approaches currentlyexist for this disease.

Like most human cancers, a small subset of neuroblastoma cases areinherited in an autosomal dominant manner (Knudson et al. (1972) Amer.J. Hum. Genet., 24:514-522; Kushner et al. (1986) Cancer, 57:1887-1893;Maris et al. (1997) Eur. J. Cancer, 33:1923-1928). A family history ofthe disease is found in about 1-2% of newly diagnosed cases, with astandardized incidence ratio of 9.7 for siblings of index cases(Friedman et al. (2005) Cancer Epidemiol. Biomarkers Prev., 14:1922-7).Neuroblastoma pedigrees show striking heterogeneity in the type oftumors that arise, with both benign and malignant forms occurring in thesame family (Maris et al. in Neuroblastoma (eds. Cheung et al.) 21-26(Springer, Berlin, Heidelberg, New York, 2005). Familial neuroblastomapatients differ from those with sporadic disease in that they arediagnosed at an earlier age and/or with multiple primary tumors,clinical characteristics that are hallmarks of cancer predispositionsyndromes. Because of the lethality of the condition prior toreproductive age, previous genetic linkage scans have been underpoweredand results difficult to replicate (Longo et al. (2007) Hum. Hered.,63:205-11; Maris et al. (2002) Cancer Res., 62:6651-6658; Perri et al.(2002) Oncogene 21:8356-60). Remarkably, neuroblastoma can occur with aspectrum of disorders related to abnormal development of neural crestderived tissues including central congenital hypoventilation syndromeand Hirschsprung disease. Missense or nonsense mutations in PHOX2B(paired-like homeobox 2B), a homeobox gene that is a master regulator ofnormal autonomic nervous system development, were recently shown topredispose to this rare field defect of the sympathicoadrenal lineagetissues (Amiel et al. (2003) Nat. Genet., 33:459-61; Mosse et al. (2004)Am. J. Hum. Genet., 75:727-30; Trochet et al. Am. J. Hum. Genet.,74:761-4). However, PHOX2B mutations explain only a small subset ofhereditary neuroblastoma, are almost exclusive to cases with associateddisorders of neural crest-derived tissues, and are not somaticallyacquired in tumors (Raabe et al. (2008) Oncogene, 27:469-76; van Limptet al. (2004) Oncogene, 23:9280-8), leaving the genetic etiology for themajority of familial neuroblastoma cases unknown.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods of detecting anincreased risk for neuroblastoma in a subject are provided. Methods ofdiagnosing and/or prognosing neuroblastoma in a subject are alsoprovided. In a particular embodiment, the method comprises obtaining abiological sample from the subject and determining whether theanaplastic lymphoma kinase (ALK) gene and/or protein is altered in thebiological sample, wherein the presence of the alteration of the ALKgene and/or protein is indicative of neuroblastoma in the subject and/orindicative of an increased risk of metastasis and/or death. In anotherembodiment, the alteration in the ALK gene is selected from the groupconsisting of an amplification the ALK copy number, presence of at leastone mutation which increases ALK activity, increased levels of ALKphosphorylation, and a translocation involving ALK which increases ALKactivity. In a particular embodiment, the ALK mutations which increaseALK activity are in the tyrosine kinase domain.

In accordance with another aspect of the instant invention, methods fortreating, inhibiting, and/or preventing (e.g., inhibiting the onset)neuroblastoma in a patient are provided. In a particular embodiment, themethods comprise the administration of at least one compositioncomprising at least one ALK inhibitor and, optionally, at least onechemotherapeutic agent. In another embodiment, the methods comprise theadministration of at least one composition comprising at least one ALKantibody and, optionally, at least one ALK inhibitor, at least one otherchemotherapeutic agent or therapy, and/or at least one GD2 antibody. Inanother embodiment, the patient is screened prior to administration ofthe composition in order to determine which ALK inhibitor is mosteffective against the particular neuroblastoma of the patient.

In yet another aspect of the invention, methods of determining whether acompound is effective for treating neuroblastoma are provided. In oneembodiment, the method comprises contacting cells comprising mutationsin ALK or an amplification of ALK encoding nucleic acid molecules, withat least one compound; and determining the ALK activity or cellviability or proliferation, wherein a reduction in ALK activity or cellviability or proliferation indicates the compound is therapeutic fortreating a neuroblastoma which comprises the mutation or amplification.

In accordance with another aspect of the instant invention, microarrayscomprising oligonucleotide probes which specifically hybridize with atleast one ALK mutant are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the eight neuroblastoma pedigrees with ALKmutations. All family members with DNA available for genotypingindicated with either wild type (wt) for ALK, or with mutation in theALK tyrosine kinase domain (R1192P, R1275Q, G1128A). Individualsaffected by neuroblastoma indicated by filled symbol.

FIG. 2A provides a schematic diagram indicating protein structure ofALK, with mutations discovered in constitutional DNAs of familial cases(germline) and primary tumors from sporadic cases (somatic) indicated.All but one sequence alteration mapped to the tyrosine kinase domain(D1091N was just N-terminal and is not indicated here). Of the threegermline mutations discovered, only the R1275Q was found in the tumorDNA samples. Conversely, the 11250T mutation discovered in the tumor setwas also present in the matched germline DNA of that patient, while allof the other mutations studied here were somatically acquired. FIG. 2Bprovides a homology model of wild-type ALK with each major subdomainindicated

(Torkamani et al. (2007) Bioinformatics, 23:2918-25; Torkamani et al.(2008) Cancer Res., 68:1675-82). FIG. 2C shows ALK mutations mapped ontohomology model (orientation different to show all mutations) with shadesindicating subdomain in which the mutation resides (e.g. the R1275Qmutation falls within the activation segment).

FIGS. 3A-3E provide representative ALK copy number alterations in fiveneuroblastoma primary tumors. Hybridization intensity reflecting copynumber for all SNPs along chromosome 2p in three primary tumors frompatients with sporadically occurring disease is shown, represented on alogarithmic scale. MYCN amplification is present in all tumors. FIG. 3Ais a regional gain (trisomy) of chromosome 2p, including the ALK locus.FIG. 3B is the focal gain of the ALK locus. FIG. 3C is the focalamplification of the ALK locus. FIGS. 3D and 3E are a complexrearrangement of the 2p locus, showing various focal amplicons,including MYCN and ALK. FIG. 4 demonstrates that ALK is highly expressedand the kinase is phosphorylated in neuroblastoma cell lines harboringactivating mutations. FIG. 4A provides a graph showing the relative ALKexpression of neuroblastoma cell lines and fetal brain determined usingthe 2-AACt method (Livak et al. (2001) Methods 25:402-8.). Statisticalsignificance was determined by unpaired T-test. FIG. 4B providesimmunoblots showing differential ALK expression in neuroblastoma celllines with phosphorylation of the tyrosine 1604 codon restricted to celllines with mutations (the wild-type lines NBEC I and NB1771 show faintphosphostaining).

FIGS. 5A-5L demonstrate that ALK knockdown results in growth inhibitionof ALK mutated or amplified neuroblastoma cell lines. FIGS. 5A-5J showcellular growth for ten neuroblastoma cell lines that were transfectedwith siRNAs against ALK or GAPDH (two negative controls and one positivecontrol not shown for clarity). The x-axis is time in hours aftertransfection, the y-axis is percent growth normalized to the siRNAagainst GAPDH. FIG. 5K provides a summary of percentage growthinhibition with ALK siRNA knockdown by ALK mutational and allelicstatus. FIG. 5L provides an immunoblot showing a time course of ALKprotein knockdown in the cell lines KELLY and SKNDZ.

FIG. 6A is a graph of a dose response curve and FIG. 6B is a graph ofthe % growth inhibition with PF066 at 333 nM.

FIG. 7 shows the expression of pALK, pAKT, pSTAT3, and pMAPK3.

FIG. 8A provides the IC₅₀ of various drugs on the neuroblastoma cellline KELLY (F1147L). FIG. 8B provides graphs of tumor volume after weeksof administration of PF'066.

FIG. 9A provides an amino acid sequence of ALK (SEQ ID NO: 5). FIG. 9Bprovides a nucleotide sequence of ALK (SEQ ID NO: 6).

FIGS. 10A and 10B demonstrate that a gain of ALK locus correlates withincreased mRNA expression. The significance of recurrent regionalgain/amplification on the p-arm of chromosome 2 for 591 tumors wasassessed using the Significance Testing for Aberrant Copy (STAC)algorithm (FIG. 10A). Significance is plotted as -log10 (P-value) in1-Mb windows along the p-arm of chromosome 2. Flat line marks thresholdfor statistical significance (adjusted P<=0.05). Frequency statistic(dark line) reveals significant recurrent focal amplification of bothMYCN (P<0.001) and ALK (P=0.026). Footprint statistic (light line)reveals large 41-Mb region of recurrent low-level gain encompassing bothMYCN and ALK (P=0.004). FIG. 10B shows that ALK mRNA expression levelsare significantly increased in tumors harboring either focal high levelamplification (P<0.0001) or low-level regional gain (P<0.0001) whencompared to tumors with no regional gain of ALK. Box and whisker plot ofrelative ALK mRNA expression is shown; lower and upper whiskersrepresent 5th and 95th percentile respectively.

FIGS. 11A and 11B show mRNA expression and constitutive phosphorylationof ALK in RPE1 cells expressing activating ALK mutations. FIG. 11Aprovides the relative ALK expression of 2 neuroblastoma cell lines (NB1and NB 1643), HTERT-RPE1 cell lines transfected with NPM-ALK and 4 ALKmutants, wild-type ALK, empty vector, and native cells, determined usingthe 2^(−ΔΔC) _(T) method (Livak et al. (2001) Methods 25:402-408). FIG.11B provides immunoblots showing differential pALK expression at Iminute and 5 minutes in the various HTERT-RPE1 cells transfected withNPM-ALK, 4 ALK mutants, wild-type ALK, empty vector and native RPE1cells.

FIG. 12 shows the varying sensitivity of different ALK aberrations toALK inhibition. Proliferation of neuroblastoma cell lines was measuredover 72 hours of incubation with PF2341066, 333 nM in DMSO using theRT-CES system. Cell lines harboring ALK amplification or mutations weresignificantly more sensitive than cell lines with normal copy number,wild type ALK (p=0.0004). In addition, cell lines harboring the R1275Qmutation were significantly more sensitive than cell lines harboringF1174L mutations (P =0.041). Inhibition of growth %=100*(cell indexvehicle-cell index treatment)/cell index control.

FIGS. 13A-13C show that in vitro growth inhibition is associated withabrogation of phosphorylation of ALK and downstream signaling proteins.Abrogation of phospho-ALK correlates to the dose where in vitroproliferation is first inhibited for all three-cell lines. NB I (wildtype amplified; FIG. 13A) and NB 1643 (R1275Q; FIG. 13B) have similargrowth inhibition. NB1 shows substantial abrogation of phosphorylationof STAT3, AKT and ERK but NB 1643 does not, indicating NB1643 may signalthrough mutation specific pathways. SHSY5Y (FIG. 13C) shows inhibitionof in vitro proliferation and abrogation of phospho-ALK at higher dosesthan NB1 and NB 1643 indicating that PF-02341066 is less able to inhibitALK signaling for this mutation.

FIGS. 14A-14E show PF-2341066 activity in vivo is associated with ALKmutations or ALK protein activation. CB17 scid mice were randomized to 4weeks of PF-2341066 100mg/kg/day via oral gavage (straight line), orvehicle (hashed line) and enrolled when xenograft volumes were 0.2-0.3cm³. Tumor volume is displayed as mean±S.E.M. The study end points forsurvival analysis were tumor volume≧1.5 cm³ or treatment related deathNB1643 (R1275Q; FIG. 14A) xenografts treated with PF-2341066 regressedcompletely by day 15 (P<0.0001). The treatment arm of SHSY5Y (F1174L;FIG. 14B) showed significant tumor growth delay (P<0.0001) and prolongedsurvival by 7.7 days (P<0.0001). NBSD (F1174L; FIG. 14C) which were moreresistant than SHSY5Y in vitro, did not show significant tumor growthdelay (P=0.3), but did prolong survival by 3.7 days (P=0.04). Twoxenograft lines were treated with WT ALK. NBEBc1 (WT; FIG. 14D) whichhas weak phospho-ALK staining showed significant delayed tumorprogression (P<0.0001), and prolongation of survival by 5.1 days(p=0.0019). By contrast, SKNAS (WT; FIG. 14E) which has low ALKexpression and no detectable pALK showed neither a delay in tumor growth(P=0.87) or prolongation of survival (P=0.70). FIGS. 15A-15D show thathomology modeling of ALK mutations predicts differential sensitivity topharmacologic inhibition. FIG. 15A provides a model of PF-02341066binding to ALK. A homology model of the PF-02341066 binding to ALK wasderived from the crystal structure of PF-02341066 bound to the kinasedomain of c-Met (PDB entry=2WGJ). Only selected side chain residues areshown. FIG. 15B provides a model of the interaction between PF-02341066and the activation loop of ALK. Van der Waals atomic surfaces aredepicted for PF-02341066 and for residues 1270-1278 of the ALK model.FIG. 15C provides a model of R1275Q mutation in ALK. Modeling predictsthat the side chain of R1275 is on the protein surface and that a R1275Qsubstitution is unlikely to result in a large destabilization ofPF-02341066 binding to ALK. FIG. 15D shows that modeling predicts lossof stabilizing protein interactions in F1174L ALK. Direct interactionsbetween F 1174, F 1245, and F1271 are predicted to stabilize the proteinconformation necessary for tight binding of PF-02341066. Substitution ofleucine at position 1174 is predicted to result in a significantdecrease in attractive interactions within this hydrophobic core.

FIG. 16 is a graph of normalized cell index of SH-Sy5Y (F1174L) cellstreated with vehicle, PF-1066, mAb30+49, or PF-1066 and mAb30+49.

FIG. 17A provides a graph showing ALK expression in 229 neuroblastomapatient tumors analyzed by Affymetrix Human Exon Array and normalizedusing quantile normalization (HRA=High risk MYCN amplifiedneuroblastoma, n=64; HRN=High risk MYCN non-amplified neuroblastoma,n=141; LR=Low risk, n=24). FIG. 17B provides representative images forimmunohistochemical staining of ALK in neuroblastoma patient tumors. ALKstaining was positive overall in 109 of 126 (86.5%) samples analyzed. 17samples showed no positive staining (upper left panel); 35 showedweak/Grade 1 staining (upper right panel); 55 showed moderate/Grade 2staining (lower left panel); and 19 showed strong/Grade 3 staining(lower right panel). FIG. 17C provides box plots showing 10thpercentile, 90th percentile, and mean ALK score by immunohistochemistryfor INSS stage (top panel) and MYCN status (bottom panel; A=MYCNamplified, N=MYCN non-amplified). **, p<0.01; *, p<0.05. FIG. 17Dprovides a representative flow cytometry analysis of NB1 cells with anALK antibody to assess cell-surface expression levels. Mean fluorescenceintensity (MFI) is shown for ALK staining (black line) and an isotypecontrol (grey line). FIG. 17E provides a graph showing the comparison offlow cytometry results for several neuroblastoma cell lines. Grey barsshow flow cytometry results for ALK cell surface staining. White barsrepresent an ALK mRNA expression ‘index’ (relative expression) measuredas ALK levels relative to HPRT1. FIG. 17F provides immunofluorescencestaining of neuroblastoma cells lines with anti-ALK demonstrates cellsurface ALK expression for NB1 (left panel) and SY5Y cells (rightpanel).

FIG. 18A provides a graph showing ALK antibody-induced growth inhibitionand ADCC of neuroblastoma cells. To measure growth inhibition uponantibody exposure, cell lines were plated in 96-well plates and treatedwith anti-ALK (mAb30 plus mAb49) or a negative control murine IgGl. Cellgrowth was monitored by Real Time Cell Electronic Sensing (RT-CES)impedance measurement. Growth inhibition of SY5Y cells treated withindicated amounts of anti-ALK as compared to control Ig. FIG. 18B showsthe indicated cell lines were treated with 10 μg/ml ALK antibody andgrowth inhibition was measured after 144 hours. FIG. 18C provides agraph showing the effect of anti-ALK antibody on RPE1 cell growth.ALK-negative RPE-1 cells were plated in 96-well plates and treated witheither 10 μg/ml anti-ALK antibody or murine immunoglobulin . Shown iscell growth for each condition as measured by RT-CES. In FIG. 18D, ADCCwas measured using an in vitro assay in which normal donor peripheralblood lymphocytes (PBL), pre-incubated overnight with IL-2, wereco-incubated for four hours with neuroblastoma cells in the presence(black line) or absence (grey line) of 1 μg/ml ALK antibody. Shown are %cytotoxicity at the indicated effector:target ratios when NB1 cells(left panel), SY5Y cells (middle panel), or ALK-negative SKNAS cells(right panel) were used as targets.

FIG. 19A shows the effect of crizotinib on cell surface ALK expression.SY5Y cells were incubated with crizotinib or vehicle, harvested andstained for cell surface ALK with mAb14. ViaProbe viability stain wasused to exclude non-viable cells. A representative flow cytometry resultis shown for SY5Y cells incubated for 72 hours with either vehicle(medium gray line) or 1000 nM crizotinib (black line). The single-peakedlight grey line represents the isotype control. FIG. 19B shows theconcentration dependence of the percent change in cell surface ALK, asmeasured by Mean Fluorescence Intensity (MFI), for cells incubated for72 hours with varying concentrations of crizotinib as compared tovehicle. FIG. 19C provides a time course of percentage change in cellsurface ALK levels (over that seen for vehicle treatment) when cellswere incubated with 1000 nM crizotinib. FIG. 19D shows a comparison ofALK antibodies for flow cytometry. SY5Y cells were treated with 1000 nMcrizotinib or vehicle, harvested 72 hours later, and stained for flowcytometry. Shown is the percent increase in ALK MFI for crizotinibversus vehicle treated cells using either the mAbl4 or mAB46 anti-ALKantibodies.

FIG. 20 shows dual antibody/TKI targeting of ALK. SY5Y cells weretreated with either 333 nM crizotinib or 10 μg/ml anti-ALK antibody(mAb30+mAb49), or both. As negative control, cells were treated withequal volumes of DMSO and 10 μg/ml IgG1. FIG. 20A shows cell growthmonitored by RT-CES, revealing clear growth inhibition by thecrizotinib/mAb combination. FIG. 20B provides an immunoblot analysis ofnative ALK protein levels (upper panel) and phospho-ALK (middle panel).β-actin levels are shown as a loading control (lower panel). FIG. 20Cshows the effect of crizotinib pre-treatment on anti-ALK antibodymediated ADCC. SY5Y cells were pre-incubated in the presence ofcrizotinib or vehicle for 48 hours, harvested, and then used as targetcells in the in vitro ADCC assay. **, p<0.01; *, p<0.05.

FIG. 21 shows the effects of an antagonist ALK antibody on crizotinibdose-response curve. SY5Y cells were treated with crizotinib at theindicated doses either alone or in combination with 10 μg/ml (total) ALKantibody mAb30 and mAb49. Cell growth was measured at day 7 usingRT-CES. FIG. 21A shows the comparison of growth inhibition formonotherapy versus dual ALK targeting: white bars represent crizotinibalone, grey bars crizotinib treatment in the presence of 10 μg/mlanti-ALK. **, p<0.01; *, p<0.05. In FIG. 21B, IC₅₀ was calculated over arange of 10 doses of crizotinib alone (circles) or crizotinib plus 10μg/ml anti-ALK (squares), yielding IC₅₀ values of 3018 nM and 1745 nMrespectively.

FIG. 22 shows the cell cycle analysis of inhibitor-treated cells. SY5Ycells were treated with 1000 nM crizotinib, 10 μg/ml antibody, both, orvehicle/IgG1, and were then harvested, fixed, stained with propidiumiodide, and analyzed by flow cytometry. FIG. 22A provides representativehistograms showing proportion of cells in sub G0/apoptosis, G0/G1, andG2/mitosis. FIG. 22B provides the quantification of flow cytometryresults. ** p<0.01; * p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

As stated hereinabove, neuroblastoma is a childhood cancer that can beinherited, but the genetic etiology was largely unknown. Here it isshown that germline mutations in the anaplastic lymphoma kinase gene(ALK) explain the majority of hereditary neuroblastomas and thatactivating mutations can also be somatically acquired. A significantlinkage signal at chromosome 2p24-23 was first identified using awhole-genome scan in neuroblastoma pedigrees. Resequencing of regionalcandidate genes identified three separate germline missense mutations inthe tyrosine kinase domain of ALK that segregated with the disease ineight separate families. Resequencing in 194 high-risk neuroblastomasamples showed somatically (only in tumor cells) acquired mutationswithin the tyrosine kinase domain in 12.4%.

Nine of the ten mutations map to critical regions of the kinase domainand were predicted to be oncogenic drivers with high probability.Mutations resulted in constitutive phosphorylation, and targetedknockdown of ALK mRNA resulted in profound growth inhibition of all celllines harboring mutant or amplified ALK, as well as 2 of 6 wild type forALK. These results demonstrate that heritable mutations of ALK are themajor cause of familial neuroblastoma, and that germline or acquiredactivation of this cell surface kinase is a tractable therapeutic targetfor this lethal pediatric malignancy

It has been predicted that neuroblastoma, like the analogous embryonalcancer retinoblastoma, would follow a two-hit model explaininghereditary and sporadic cases (Knudson et al. (1972) Amer. J. Hum.Genet., 24:514-522). This model has proven to be correct for themajority of childhood and adult hereditary cancers and thesusceptibility genes are typically tumor suppressors where the two hitsare sequential inactivation of both alleles. Discovery of heritablemutations in oncogenes as the etiology of multiple endocrine neoplasia 1cancers (RET), papillary renal carcinoma (MET) and gastrointestinalstromal tumors (KIT) challenged this paradigm, but it is now clear thatsomatically acquired duplication or amplification of the mutant alleleprovides the second hit (Vogelstein et al. (2004) Nat. Med., 10:789-99).It is shown herein that heritable mutations in ALK are the cause of themajority of hereditary neuroblastoma cases, providing the first exampleof a pediatric cancer arising due to mutations in an oncogene. Takentogether with the recent report that common variations at chromosomeband 6p22 predispose to the development of sporadic neuroblastoma (Mariset al. (2008) N. Engl. J. Med., 358:2585-93), the genetic etiology ofthis disease is now being defined. The discovery of highly penetrantheritable ALK mutations as the cause of hereditary neuroblastoma are ofimmediate relevance to patients with a family history as screening withnoninvasive techniques such as ultrasonography and measurement ofurinary catecholamine metabolites should likely be implemented forunaffected children carrying an ALK mutation. ALK is an orphan tyrosinekinase transmembrane receptor with homology to neurotrophin receptorsand the MET oncogene. Expression is restricted to the developing nervoussystem with a postulated role in participating in the regulation ofneuronal differentiation (Iwahara et al. (1997) Oncogene, 14:439-49). Itis now clear that many human cancers activate ALK signaling by creatingunique oncogenic fusions of ALK with a variety of partners throughchromosomal translocation events (Chiarle et al. (2008) Nat. Rev.Cancer, 8:11-23). Previous work had shown that a substantial percentageof human-derived neuroblastoma cell lines express ALK transcripts andALK protein (Lamant et al. (2000) Am. J. Pathol., 156:1711-21), but nodefinitive role for this oncogene had been proven (Osajima-Hakomori etal. (2005) Am. J. Pathol., 167:213-22; Motegi et al. (2004) J. CellSci., 117:3319-29; Miyake et al. (2002) Oncogene, 21:5823-34; Dirks etal. (2002) Int. J. Cancer, 100:49-56). ALK has recently been identifiedas a molecular target in neuroblastoma through a screen of human cancercell lines with pharmacologic antagonists of the ALK kinase domain(McDermott et al. (2008) Cancer Res., 68:3389-95). The data hereinprovides the first evidence for oncogenic activation of ALK via mutationof the kinase domain, and these data provide the genetic basis for theobservation of sensitization to ALK kinase inhibition. In addition, thediscoveries in neuroblastoma may lead to future resequencing efforts inother malignancies, especially those where oncogenic fusion proteinshave recently been discovered. The data presented here clearly establishALK as critical neuroblastoma oncogene and should increase efforts toidentify the ligand for this receptor and understand if ALK-mediatedsignaling can be activated by mechanisms other than direct mutationand/or amplification of ALK alleles. Finally, receptor tyrosine kinasesprovide tractable targets for pharmacologic inhibition, and allows fortherapeutic strategies aimed at inhibiting ALK-mediated signaling.

In accordance with the instant invention, methods of identifying,determining an increased risk for, diagnosing, and/or prognosing acancer in a patient are provided, wherein the method comprisesdetermining the level/activity of ALK. In a particular embodiment, thecancer is neuroblastoma. In another embodiment, the cancer has beencharacterized as having an ALK translocation (e.g., an ALK translocationwherein the resultant ALK fusion protein is constitutively active). Suchcancers include, without limitation, lymphomas, non-Hodgkin's lymphoma,anaplasic large cell lymphoma, inflammatory myofibroblastic tumors, andnon-small-cell lung cancer. The methods may further comprise obtaining abiological sample from the subject. In a particular embodiment, thebiological sample is tumor tissue or blood.

In one embodiment, the method comprises determining the presence of atleast one mutation in ALK, particularly one which leads to increasedactivity of ALK (e.g., increased kinase activity). According to oneembodiment, the mutation is within the kinase domain. In anotherembodiment, at least one amino acid at position P36, P157, V198, G640,L684, G718, D993, L1204, 11170, A1200, L1204, F1245, G1128, R1192,R1275, D1091, M1166, 11171, F1174, F1245, 11250, and those set forth inTables 2A and 2B (e.g., TI 151, L1196, R259, M770, E1407, E1433, R1464,G1494, A1553) is altered (mutated). In another embodiment, at least oneamino acid at position G1128, R1192, R1275, D1091, M1166,11171, F1174,F1245, and 11250 is altered (mutated). In still another embodiment, atleast one amino acid at position G1128, R1192, L1204, 11250, R1275, andthose set forth in Tables 2A and 2B (e.g., P36, V198, R259, G640, D993,E1407, and A1553); particularly, at least one amino acid at position GI128, R1192, and R1275 is altered (mutated; particularly those set forthbelow) when the germline is examined (e.g., when the biological sampleis not tumor tissue). In one embodiment, at least one amino acid atposition P36, P157, V198, G640, L684, G718, D993, L1204, 11170, A1200,L1204, G1128, R1192, R1275, D1091, M1166, 11171, F1174, F1245, 11250 andthose set forth in Tables 2A and 2B (e.g., T1151, 11170, L1196, R259,M770, E1407, E1433, R1464, G1494, A1553); particularly, at least oneamino acid at position P36, P157, V 198, G640, L684, G718, G718, D993,L1204, 11170, A1200, L1204, F1245, R1275, D1091, M1166, 11171, F1174,F1245, and 11250; particularly, at least one amino acid at positionR1275, D1091, M1166, 11171, F1174, F1245, and 11250 is altered (mutated;particularly those set forth below) when somatic mutations are examined(e.g., when the biological sample is tumor tissue/cells). In anotherembodiment, the mutation may be a nonconservative amino acidsubstitution. In still another embodiment, the ALK comprises at leastone mutation selected from the group consisting of P36S, P157S, V198M,G640R, L684M, G718F, G718S, D993G, L1204F, I117OS, A1200V, L1204F,F1245I, G1128A, R1192P, R1275Q, D1091N, M1166R, I1171N, F1174I, F1174L,F1245C, F1245V, I1250T, and those set forth in Tables 2A and 2B (e.g.,T1151M, I1170S, F1174C, L1196M, F1245I, R259H, M770I, E1407K, E1433del,R1464G, G1494R, and A1553P). In another embodiment, at least onemutation selected from the group consisting of G1128A, R1192P, R1275Q,D1091N, M1166R, I1171N, F1174I, F1174L, F1245C, F1245V, and I1250T. Inyet another embodiment, at least one of the mutations is to amino acidR1275 and/or F1174, particularly at least one of R1275Q, F1174I, andF1174L. The presence of at least one of the above mutations isindicative of neuroblastoma or at least an increased risk of developingneuroblastoma in the patient. The presence of at least one of the abovemutations is also indicative of a poor prognosis with increased risk ofmetastasis and higher risk of death. While the above mutations can bedetected by sequencing the ALK protein in a biological sample obtainedfrom a subject, it is preferred that the nucleic acid molecule encodingALK is examined in the instant methods (after obtaining (e.g.,isolating) from a biological sample from a subject). The ability todetect the above mutations in a nucleic acid molecule/protein are wellknown in the art and include, without limitation, sequencing, PCR (e.g.,real time PCR; e.g., with mutation specific primers; optionally withsubsequent sequencing or hybridization), hybridization techniques (e.g.,with mutation specific probes (probes which specifically bind a mutatedALK to the exclusion of wild-type ALK); e.g., microarrays, Southern,Northern), and antibodies (e.g., those specific for at least onemutant). In yet another embodiment, the methods of the instant inventioncomprise determining the ALK copy number in the cells of the biologicalsample obtained from a subject. A gain or amplification in the ALK copynumber compared to normal human cells is indicative of neuroblastoma orat least an increased risk of developing neuroblastoma in the patient.The increased ALK copy number is also indicative of a poor prognosiswith increased risk of metastasis and higher risk of death.

In still another embodiment, the methods of the instant inventioncomprise determining if the ALK is phosphorylated. In one embodiment,the ALK is phosphorylated to greater levels than ALK from a normal human(i.e., one that does not have cancer, particularly neuroblastoma). TheALK may be phosphorylated at positions that are not phosphorylated innormal humans and/or phosphorylated to greater levels (greaterfrequency) than ALK in normal humans. For example, as describedhereinbelow, the constitutive phosphorylation (increased levels ofphosphorylation compared to normal humans) of tyrosine at position 1604of ALK is indicative of neuroblastoma or at least an increased risk ofdeveloping neuroblastoma in the patient. The increased ALKphosphorylation is also indicative of a poor prognosis with increasedrisk of metastasis and higher risk of death.

According to another aspect of the instant invention, the above methodsfor identifying, diagnosing, or prognosing cancer (particularlyneuroblastoma) in a patient, further comprises identifying mutations inthe phox2B gene/protein (e.g., 676delG) or amplification of the phoX2Bgene/protein (see, e.g., Mosse et al. (2004) Am. J. Hum. Genet.,75:727-730; Rabbe et al. (2008) Oncogene 27:469-476).

In accordance with another aspect of the instant invention, methods fortreating cancer, particularly a neuroblastoma, in a patient areprovided, where the method comprises the administration of a compositioncomprising at least one ALK inhibitor (e.g., an inhibitor of ALK kinaseactivity) and at least one pharmaceutically acceptable carrier. Themethod may further comprise determining the particular ALK alteration ofthe patient prior to administration (see above) and administering theALK inhibitor most effective for the ALK alteration identified (seebelow). Examples of ALK inhibitors include, without limitation, ALKsiRNA and/or antisense molecules, small molecule inhibitors, PF-02341066(Pfizer), TAE684 (Novartis), and CEP-14083 (Cephalon). The methods mayalso comprise the administration of an antibody (or fragment thereof)specific for ALK (e.g., monoclonal antibodies). In a particularembodiment, the antiboilies are specific for the extracellular domain ofALK (e.g., the extracellular domain that remains after proteolyticcleavage from the 220 kDa to 140 kDa species). The antibodies may beadministered separately (before, after, or at the same time as the ALKinhibitor) or in the same composition. The methods may also comprise theadministration of at least one other chemotherapeutic agent and/or beadministered in coordination with another chemotherapeutic agent ortherapy (e.g., chemotherapy). The chemotherapeutic agent may beadministered separately (before, after, or at the same time as the ALKinhibitor) or in the same composition. The compositions may beadministered by any method such as, for example, intravenous injectioninto the blood stream, oral administration, or by subcutaneous,intramuscular or intraperitoneal injection. As stated hereinabove, themethods may also further comprise first screening the subject todetermine the ALK mutation (including amplification of copy number)present in the subject as described hereinabove and selecting theappropriate ALK inhibitor for the identified mutation to administer tothe patient (see below).

In accordance with another aspect of the instant invention, methods ofidentifying an agent which is therapeutic for the treatment ofneuroblastoma are provided. In a particular embodiment, the methodcomprises contacting cells comprising mutations in ALK or anamplification of ALK with at least one agent and determining the ALKactivity, wherein a reduction in ALK activity indicates the agent is atherapeutic agent for treating neuroblastoma. In another embodiment, themethod comprises contacting cells comprising mutations in ALK or anamplification of ALK with at least one agent and determining the abilityof the agent to inhibit proliferation of the cells (e.g., determiningIC₅₀), wherein a reduction in proliferation indicates the agent is atherapeutic agent for treating a neuroblastoma characterized by the ALKmutation of the cells.

In accordance with another aspect of the present invention, microarraysfor detecting ALK and/or the ALK mutants described hereinabove areprovided. In a particular embodiment, the microarray comprisesantibodies specific for ALK and/or the ALK mutants describedhereinabove. In a preferred embodiment, the microarray comprisesoligonucleotide probes which recognize ALK and/or the ALK mutantsdescribed hereinabove. The microarrays may comprise oligonucleotideprobes which specifically hybridize with at least 2, at least 5, atleast 10, or all of the above ALK mutants. In a particular embodiment,the microarray comprises oligonucleotide probes wherein each probe (orcoordinate on the microarray) specifically hybridizes with a single ALKmutant (e.g., a single nucleotide change, a single amino acid changeencompassing all codons of the amino acid change, or all changes to asingle amino acid position). In a particular embodiment, theoligonucleotide probe is completely complementary to ALK (e.g., SEQ IDNO: 6) except for the mutation. In yet another embodiment, theoligonucleotide is about 10, 15, 20, 25, or 30 to about 40, 50, 75, or100 nucleotides in length. In a particular embodiment, theoligonucleotide probes span an ALK mutant above, particularly so thatthe mutation is in the middle of the probe (e.g., within the middlethird of the probe). In another embodiment, the microarray furthercomprises probes specific for wild-type ALK and/or PHOX2B (wild-typeand/or mutant). In another embodiment, the microarray is containedwithin kit further comprising instruction material and, optionally, atleast one positive control (a nucleic acid molecule recognized by anoligonucleotide probe of the microarray) and/or at least one negativecontrol (a nucleic acid molecule not recognized by an oligonucleotideprobe of the microarray).

Therapeutic methods and compositions

Recent studies have shown that crizotinib, a dual Met/ALK TKI, inducesremarkable tumor regression in NSCLC patients harboring ALKtranslocations (Kwak et al. (2010) N. Engl. J. Med., 363:1693-703).Crizotinib is also currently in early-phase clinical trial testing inpatients with neuroblastoma. However, preclinical studies have shownthat the impact of crizotinib is highly dependent on ALK copy number andgenotype. For instance, cell lines harboring the F1174L mutation, thesecond most common ALK mutation seen in neuroblastoma tumors, aresignificantly more resistant to crizotinib than those harboring the mostcommon mutation, R1275Q (George et al. (2008) Nature 455:975-8; Sasakiet al. (2010) Cancer Res.). Moreover, resistance mutations in oncogenicALK fusions have already emerged in early studies with crizotinib(Sasaki et al. (2010) Cancer Res.; Engelman et al. (2008) Curr. Opin.Genet. Dev., 18:73-9; Choi et al. (2010) N. Engl. J. Med., 363:1734-9).These findings underline a need for developing additional therapeuticoptions in neuroblastoma, where well over 50% of high-risk patients willeventually die of their disease despite major recent therapeuticadvances (Maris, J.M. (2010) N. Engl. J. Med., 362:2202-11; Haupt et al.(2010) J. Clin. Oncol., 28:2331-8). One such option is immunotherapy.Herein, it is shown that ALK antibodies inhibit the growth ofneuroblastoma cell lines. Further, the utility of combining ALKantibodies with TKIs is demonstrated as an important therapeuticstrategy in neuroblastoma.

As taught herein, ALK is a receptor tyrosine kinase aberrantly expressedin neuroblastoma, a devastating pediatric cancer of the sympatheticnervous system. Germline and somatically acquired ALK aberrations induceincreased autophosphorylation, constitutive ALK activation, andincreased downstream signaling. Thus, ALK is a tractable therapeutictarget in neuroblastoma which can be susceptible to both small moleculetyrosine kinase inhibitors and therapeutic antibodies. Small moleculeinhibitors of ALK are available and more are currently being developedin the clinic, but common ALK mutations in neuroblastoma appear to showde novo insensitivity, arguing that complementary approaches must bedeveloped. It is shown herein that antibody targeting of ALK is atherapeutically relevant strategy for neuroblastoma patients likely tohave ALK-positive tumors. An antagonistic ALK antibody is shown hereinto inhibit cell growth and induces in vitro antibody-dependent cellularcytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity wasinduced in cell lines harboring either wild-type or mutated forms ofALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitorcrizotinib sensitized cells to antibody-induced growth inhibition bypromoting cell surface accumulation of ALK and thus increasing theaccessibility of antigen for antibody binding. These data clearly showthat ALK-targeted immunotherapy is a strong therapeutic strategy forneuroblastomas with mutated or wild-type ALK.

Approaches for therapeutically targeting RTKs include monoclonalantibodies and small molecule tyrosine kinase inhibitors (TKIs), both ofwhich have led to dramatic increases in survival and time to progressionin certain types of cancer (Zhang (2009) Nat. Rev. Cancer 9:28-39;Weiner et al. (2010) Nat. Rev. Immunol., 10:317-27). For example, thetrastuzumab antibody was approved for treatment of HER2-overexpressingbreast cancer over 10 years ago, and is thought to have multiplemechanisms of action including blockade of aberrant signaling andantibody-dependent cellular cytotoxicity (ADCC) (Hudis, C. A. (2007) N.Engl. J. Med., 357:39-51). Similarly, the epidermal growth factorreceptor (EGFR) antibody cetuximab inhibits binding of activatingligands and induces ADCC (Kurai et al. (2007) Clin. Cancer Res.,13:1552-61). Clinical activity of TKIs that inhibit HER2 and EGFR hasbeen amply demonstrated. Moreover, these TKIs have been used with HER2-and EGFR-targeted antibodies in breast and lung cancer, respectively(Scaltriti et al. (2009) Oncogene 28:803-14; Regales et al. (2009) J.Clin. Invest., 119:3000-10; Xia et al. (2005) Oncogene 24:6213-21).

As stated herein, a recent phase 3 clinical trial using antibodiesagainst the disialoganglioside GD2, which is almost uniformly expressedon neuroblastoma cell surfaces, has shown the promise of intensivetargeted immunotherapy in neuroblastoma (Yu et al. (2010) N. Engl. J.Med., 363:1324-34). It is shown herein that ALK RTK is also expressed inthe vast majority of neuroblastoma cases, indicating that it representsanother tractable immunotherapy target. Moreover, like GD2, ALKexpression is largely limited to tumor tissue (Iwahara et al. (1997)Oncogene 14:439-49), making it an ideal target for immunotherapy whileminimizing the risk of cytotoxicity in non-malignant tissue of patientstreated with ALK antibodies.

Like the GD2 antibody, and several clinically successful antibodies suchas trastuzumab and cetuximab, it was found herein that an ALK antibodycan mediate ADCC of ALK-positive neuroblastoma cells. In the in vitroADCC system, neuroblastoma cells were killed when treated with 1 μg/mlanti-ALK antibody, a lower dose than the 10 μg/ml clinically achieved intrastuzumab-treated breast cancer patients (Baselga et al. (1996) J.Clin. Oncol., 14:737-44), and well below the trough plasma antibodyconcentrations achieved in two phase I studies of lung cancer patientstreated with cetuximab (Baselga et al. (2000) J. Clin. Oncol.,18:904-14; Robert et al. (2001) J. Clin. Oncol., 19:3234-43). Theexperiments were not designed to determine the optimal ADCC dose, butnonetheless show that antibody targeting of ALK is an importanttherapeutic avenue based on its ADCC effects alone. Additional studiesmay be performed to determine the minimum ALK antibody dose at whichADCC can be induced, the range of effective concentrations, and thedependence of ADCC sensitivity on ALK expression level and genotype.

Unlike GD2 antibodies, those that target ALK can bind and inhibit anoncogenic growth factor receptor—permitting an additional immunecell-independent component of its inhibitory mechanism, throughinactivation of a constitutively activated receptor. ALK overexpressionor mutation leads to its hyper-activation, autophosphorylation andelevated down-stream signaling (Osajima-Hakomori et al. (2005) Am. J.Pathol., 167:213-22), as also seen for HER2 and EGFR in breast and lungcancer (Yarden et al. (2001) Nat. Rev. Mol. Cell Biol., 2:127-37).Indeed, the oncogenic role of full-length ALK in neuroblastoma wasoriginally discovered when activating mutations in ALK, which occur inroughly 8% of patient tumors, were discovered in the germline ofpatients with the hereditary form of neuroblastoma, and weresubsequently found to be somatically acquired (George et al. (2007) PLoSOne 2:e255; Janoueix-Lerosey et al. (2008) Nature 455:967-70; Mosse etal. (2008) Nature 455:930-5; Chen et al. (2008) Nature 455:971-4). Ithas also been shown that whereas native ALK expression levels predictimproved survival, high levels of ALK expression (which promotes itsactivation) are associated with decreased survival (see also De Brouweret al. (2010) Clin. Cancer Res., 16:4353-62). Thus, ALKsignaling—enhanced by mutation or overexpression—appears to play animportant role in initiation and/or maintenance of neuroblastoma. ALKinhibition with the TKI crizotinib is likely to be an important tool intreating ALK-dependent neuroblastoma, but it is already clear thatcertain ALK-activating mutations reduce sensitivity to this drug.Antagonistic ALK antibodies provide a superior approach to ALKinhibition. It is shown herein that an antagonist ALK antibody inhibitsgrowth of neuroblastoma cells over a range of doses, all of which arebelow (or equal to) those reported to be clinically achievable inpatients with trastuzumab or cetuximab. The ALK antibody inhibits growthin neuroblastoma cell lines that harbor either amplified/over expressedALK (NB1) or ALK with the most common constitutively activatingmutations (R1275Q in 1643 cells, and F1174L in SY5Y cells). Notably,SY5Y cells are relatively resistant to TKI-targeting of ALK, but respondto antibody inhibition. ALK antibody therapy is, therefore, relevant forpatient tumors exhibiting a broad range of ALK expression levels andaberrations.

It is also demonstrated herein that dual targeting of ALK with bothantibody and TKI therapeutics produces superior results. Dual targetinghas been used with other oncogenic RTKs (Scaltriti et al. (2009)Oncogene 28:803-14; Xia et al. (2005) Oncogene 24:6213-21; Johns et al.(2003) Proc. Natl. Acad. Sci., 100:15871-6; Konecny et al. (2006) CancerRes., 66:1630-9; Matar et al. (2004) Clin. Cancer Res., 10:6487-501).For example, for NSCLC expressing erlotinib-resistant EGFR^(T790M), suchdual targeting induced tumor regression (Regales et al. (2009) J. Clin.Invest., 119:3000-10). For ALK-expressing neuroblastoma cell lines,crizotinib treatment enhances cell surface expression of ALK, leadingboth to enhanced ADCC and elevated immune cell-independent growthinhibition and cytotoxicity. For F1174L-expressing SY5Y cells, forexample, combined antibody/TKI treatment led to almost complete growthinhibition, and induced a significantly higher level of apoptosis thaneither crizotinib or antibody alone. Importantly, the antibody/TKIcombination therapy demonstrated efficacy at low (10 nM) doses ofcrizotinib and antibody reduced the IC₅₀ for crizotinib treatment byhalf. This shows that dual ALK targeting is a relevant therapeuticstrategy for decreasing the dose-dependent toxicities associated withTKI therapy and can delay or prevent TKI resistance.

In accordance with the instant invention, methods for inhibiting,reducing the progression of, and/or treating cancer, particularly aneuroblastoma, in a subject (e.g., human or animal) are provided. Theneuroblastoma may be resistant to an ALK inhibitor (e.g., crizotinib).

In a particular embodiment, the methOd comprises the administration ofat least one ALK antibody to a subject. In a particular embodiment, theantibody is immunologically specific for the extracellular domain of ALK(e.g., the extracellular domain that remains after proteolytic cleavagefrom the 220 kDa to 140 kDa species, or amino acids 19-1038). ALK aminoacid and nucleotide sequences are provided, e.g., in FIG. 9 as well asU.S. Pat. No. 5,770,421; GenBank Gene ID: 238; and GenBank AccessionNos. NM 004304.4 and NP 004295.2. Examples of ALK antibodies areprovided, for example, in Moog-Lutz et al. (.I. Biol. Chem.,280:26039-26048), Bernard-Pierrot et al. (J. Biol. Chem. (2002)277:32071-32077), U.S. Patent Application Publication No. 2008/0118512,U.S. Patent Application Publication No. 2001/0021505, U.S. Patent5,770,421; Motegi et al. (J. Cell Sci. (2004) 117:3319-29), and Mazot etal. (Oncogene (2011) doi:10.1038/onc.2010.595).

Anti-ALK antibody of the instant invention may be modified. For example,the antibodies may be humanized to reduce immunogenicity (Jones et al.(1986) Nature 321:522-5), de-fucosylated to maximize ADCC (Niwa et al.(2005) Clin. Cancer Res., 11:2327-36), and/or conjugated toimmunostimulatory cytokines such as IL-2 or cytotoxic agents (Hughes, B.(2010) Nat. Rev. Drug Discov., 9:665-7). In a particular embodiment, theanti-ALK antibody is operably linked (e.g., coupled or conjugated) toreagents that induce cell death. For example, the antibody may be linkedto a cytotoxic molecule, a radioisotope, drug, and/or a chemotherapeuticagent. Cytotoxic molecules include, without limitation, complement(e.g., mouse, rat, rabbit, guinea pig, cow, horse, and human),nanoparticles and nanotubes (e.g., heat sensitive carbon nanocrystals;see e.g., Chakravarty et al. (2008) PNAS 105:8697-8702) and Cho et al.(2008) Clin. Cancer Res., 14:1310-1316), cytoxic antibiotics (e.g.,calicheamicin), cationic amphipathic lytic peptides (e.g., KLA and PTP(prostate-specific membrane antigen-targeting peptide)), radionuclides,and toxins. Toxins can be derived from various sources, such as plants,bacteria, animals, and humans or be synthetic toxins (drugs), andinclude, without limitation, saprin, ricin (e.g., ricin A), abrin,ethidium bromide, diptheria toxin, Pseudomonas exotoxin, PE40, PE38,saporin, gelonin, RNAse, peptide nucleic acids (PNAs), ribosomeinactivating protein (RIP) type-1 or type-2, pokeweed anti-viral protein(PAP), bryodin, momordin, chemotherapeutic agents, and bouganin.Radionuclides (radioisotopes) of the instant invention include, withoutlimitation, positron-emitting isotopes and alpha-, beta-, gamma-, Auger-and low energy electron-emitters. In a particular embodiment, theradionuclides are alpha-emitters or auger-emitters. The radioisotopesinclude, without limitation: ¹³N, 18F, ³²P, ⁶⁴Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga,⁶⁷Cu, ⁷⁷Br, ^(80m)Br, ⁸²Rb, ⁸⁶Y, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ^(99m)Tc, ¹⁰³Ru,¹⁰⁵Ru, ¹¹¹In, ^(113m)In, ¹¹³Sn, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹²³I,¹²⁴I, ¹²⁵I, ¹²⁶I, ¹³¹I, ¹³³I, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁷⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,^(195m)Hg, ²¹¹At, ²¹²Bi, ²¹³Bi, ²¹³Bi, and ²²⁵Ac. In yet anotherembodiment, the radionuclide containing molecule can be administeredwith a radiosensitizer.

In a particular embodiment, the method further comprises theadministration of at least one ALK inhibitor (e.g., an inhibitor of ALKkinase activity) to the subject. The ALK inhibitor may be administeredbefore, after, and/or simultaneously (e.g., in the same composition orin different compositions) with the ALK antibody. In a particularembodiment, the ALK inhibitor is a small molecule inhibitor (e.g., anATP-competitive, small-molecule inhibitor of the receptor tyrosinekinase). Examples of ALK inhibitors include, without limitation,crizotinib (PF-02341066, Pfizer), LDK378 (Novartis), TAE684 (Novartis),CEP-14083 (Cephalon, Frazer, Pa.), CEP-37440 (Cephalon), CEP-28122(Cephalon), CEP-14513 (Cephalon), AF802 (Chugai (Japan), Roche), AP26113(Ariad; Cambridge, Mass.), and the compounds in U.S. Pat. Nos.7,601,716; 7,910,585; 7,893,074; and 7,671,063, and U.S. PatentApplication Publication Nos. 2009/0131436, 2009/0221555, and2009/0286778. In a particular embodiment, the ALK inhibitor iscrizotinib.

The methods may also comprise the administration of at least one otherchemotherapeutic agent and/or be administered in coordination withanother chemotherapeutic agent or therapy (e.g., chemotherapy,radiation, etc.). In a particular embodiment, disialoganglioside GD2antibodies (Yu et al. (2010) N. Engl. J. Med., 363:1324-34) are alsoadministered. The chemotherapeutic agent may be administered separately(before, after, or at the same time as the ALK inhibitor and/or ALKantibody) or in the same composition. As stated hereinabove, the methodsmay also further comprise first screening the subject to determine theALK mutation (including amplification of copy number) present in thesubject as described hereinabove and selecting the appropriate ALKinhibitor and/or antibody for the identified mutation to administer tothe patient.

Compositions comprising the above ALK therapeutics are also encompassedby the instant invention. In a particular embodiment, the compositioncomprises at least one ALK antibody and at least one pharmaceuticallyacceptable carrier. The composition may further comprise at least oneALK inhibitor, chemotherapeutic agent, and/or at least one GD2 antibody.In yet another embodiment, kits comprising at least one of the abovecompositions are encompassed by the instant invention. For example, thekit may comprise a first composition comprising at least one ALKantibody and at least one carrier and a second composition comprising atleast one ALK inhibitor and at least one carrier.

The compositions described herein will generally be administered to apatient as a pharmaceutical preparation. The term “patient” as usedherein refers to human or animal subjects. These compositions may beemployed therapeutically, under the guidance of a physician.

The compositions of the instant invention may be conveniently formulatedfor administration with any pharmaceutically acceptable carrier(s). Forexample, the agents may be formulated with an acceptable medium such aswater, buffered saline, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), dimethylsulfoxide (DMSO), oils, detergents, suspending agents or suitablemixtures thereof. The concentration of the agents in the chosen mediummay be varied and the medium may be chosen based on the desired route ofadministration of the pharmaceutical preparation. Except insofar as anyconventional media or agent is incompatible with the agents to beadministered, its use in the pharmaceutical preparation is contemplated.

The dose and dosage regimen of compositions according to the inventionthat are suitable for administration to a particular patient may bedetermined by a physician considering the patient's age, sex, weight,general medical condition, and the specific condition for which thecomposition is being administered and the severity thereof. Thephysician may also take into account the route of administration, thepharmaceutical carrier, and the composition's biological activity.

Selection of a suitable pharmaceutical preparation will also depend uponthe mode of administration chosen. For example, the compositions of theinvention may be administered intravenously. In this instance, apharmaceutical preparation comprises the agents dispersed in a mediumthat is compatible with intravenous injection.

Compositions of the instant invention may be administered by any method.For example, the compositions of the instant invention can beadministered, without limitation, parenterally, subcutaneously, orally(e.g., liquid or pill/capsule/tablet form), topically, pulmonarily,intravenously, intraperitoneally, intrathecally, epidurally,intramuscularly, intradermally. In a particular embodiment, thecompositions are administered intravenously or orally. Pharmaceuticalpreparations for injection and oral administration are known in the art.If injection is selected as a method for administering the composition,steps must be taken to ensure that sufficient amounts of the moleculesreach their target cells to exert a biological effect.

Pharmaceutical compositions containing an agent of the present inventionas the active ingredient in intimate admixture with a pharmaceuticallyacceptable carrier can be prepared according to conventionalpharmaceutical compounding techniques. The carrier may take a widevariety of forms depending on the form of preparation desired foradministration, e.g., intravenous.

A pharmaceutical preparation of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical preparation appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of active ingredientcalculated to produce the desired effect in association with theselected pharmaceutical carrier. Procedures for determining theappropriate dosage unit are well known to those skilled in the art.Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unitfor the administration of compositions of the instant invention may bedetermined by evaluating the toxicity of the molecules or cells inanimal models. Various concentrations of agents in pharmaceuticalpreparations may be administered to mice, and the minimal and maximaldosages may be determined based on the beneficial results and sideeffects observed as a result of the treatment. Appropriate dosage unitmay also be determined by assessing the efficacy of the agent treatmentin combination with other standard drugs. The dosage units of thecompositions may be determined individually or in combination with eachtreatment according to the effect detected.

The pharmaceutical preparation comprising the agents of the instantinvention may be administered at appropriate intervals, for example, atleast once or twice a day or more until the pathological symptoms arereduced or alleviated, after which the dosage may be reduced to amaintenance level. The appropriate interval in a particular case wouldnormally depend on the condition of the patient.

Definitions

As used herein, a “biological sample” refers to a sample of biologicalmaterial obtained from a subject, preferably a human subject, includinga tissue, a tissue sample, a cell sample, a tumor sample, and abiological fluid, e.g., blood or urine. A biological sample may beobtained in the form of, e.g., a tissue biopsy, such as, an aspirationbiopsy, a brush biopsy, a surface biopsy, a needle biopsy, a punchbiopsy, an excision biopsy, an open biopsy, an incision biopsy and anendoscopic biopsy.

As used herein, “diagnose” refers to detecting and identifying a diseasein a subject. The term may also encompass assessing or evaluating thedisease status (progression, regression, stabilization, response totreatment, etc.) in a patient known to have the disease.

As used herein, the term “prognosis” refers to providing informationregarding the impact of the presence of cancer (e.g., as determined bythe diagnostic methods of the present invention) on a subject's futurehealth (e.g., expected morbidity or mortality, the likelihood of gettingcancer, and the risk of metastasis). In other words, the term“prognosis” refers to providing a prediction of the probable course andoutcome of a cancer or the likelihood of recovery from the cancer.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc.

The phrase “effective amount” refers to that amount of therapeutic agentthat results in an improvement in the patient's condition.“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative(e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid,sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80),emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), water, aqueoussolutions, oils, bulking substance (e.g., lactose, mannitol), excipient,auxilliary agent or vehicle with which an active agent of the presentinvention is administered. Suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin (MackPublishing Co., Easton, Pa.); Gennaro, A. R., Remington: The Science andPractice of Pharmacy, 20th Edition, (Lippincott, Williams and Wilkins),2000; Liberman, et al., Eds., Pharmaceutical Dosage Forms, MarcelDecker, New York, N.Y., 1980; and Kibbe, et al., Eds., Handbook ofPharmaceutical Excipients (3rd Ed.), American PharmaceuticalAssociation, Washington, 1999.

As used herein, a “conservative” amino acid substitution/mutation refersto substituting a particular amino acid with an amino acid having a sidechain of similar nature (i.e., replacing one amino acid with anotheramino acid belonging to the same group). A “non-conservative” amino acidsubstitution/mutation refers to replacing a particular amino acid withanother amino acid having a side chain of different nature (i.e.,replacing one amino acid with another amino acid belonging to adifferent group). Groups of amino acids having a side chain of similarnature are known in the art and include, without limitation, basic aminoacids (e.g., lysine, arginine, histidine);

acidic amino acids (e.g., aspartic acid, glutamic acid); neutral aminoacids (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan); amino acids having a polar sidechain (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine); amino acids having a non-polar side chain (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); amino acids having an aromatic side chain(e.g., phenylalanine, tryptophan, histidine); amino acids having a sidechain containing a hydroxyl group (e.g., serine, threonine, tyrosine),and the like.

As used herein, the term “amplification” when used in reference to copynumber refers to the condition in which the copy number of a nucleicacid sequence is greater than the copy number of a control sequence. Inother words, amplification indicates that the ratio of a particularnucleic acid sequence is greater than 1:1 when compared to a controlsequence (e.g., 1.1:1, 1.2:1, or 1.3:1).

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains about 10-100,about 10-50, about 15-30, about 15-25, about 20-50, or more nucleotides,although it may contain fewer nucleotides. The probes herein may beselected to be complementary to different strands of a particular targetnucleic acid sequence. This means that the probes must be sufficientlycomplementary so as to be able to “specifically hybridize” or annealwith their respective target strands under a set of pre-determinedconditions. Therefore, the probe sequence need not reflect the exactcomplementary sequence of the target, although they may. For example, anon-complementary nucleotide fragment may be attached to the 5′ or 3′end of the probe, with the remainder of the probe sequence beingcomplementary to the target strand. Alternatively, non-complementarybases or longer sequences can be interspersed into the probe, providedthat the probe sequence has sufficient complementarity with the sequenceof the target nucleic acid to anneal therewith specifically.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such asappropriate temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically about 10-25 or more nucleotides inlength. The primer must be of sufficient complementarity to the desiredtemplate to prime the synthesis of the desired extension product, thatis, to be able to anneal with the desired template strand in a mannersufficient to provide the 3′ hydroxyl moiety of the primer inappropriate juxtaposition for use in the initiation of synthesis by apolymerase or similar enzyme. It is not required that the primersequence represent an exact complement of the desired template. Forexample, a non-complementary nucleotide sequence may be attached to the5′ end of an otherwise complementary primer.

Alternatively, non-complementary bases may be interspersed within theoligonucleotide primer sequence, provided that the primer sequence hassufficient complementarity with the sequence of the desired templatestrand to functionally provide a template-primer complex for thesynthesis of the extension product. Polymerase chain reaction (PCR) hasbeen described in U.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965,188,the entire disclosures of which are incorporated by reference herein.

With respect to single stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence. Appropriate conditions enabling specific hybridization ofsingle stranded nucleic acid molecules of varying complementarity arewell known in the art.

For instance, one common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified sequence homology is set forth below (Sambrooket al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63 (% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C.with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated Tm of the hybrid. Washconditions should be as stringent as possible for the degree of identityof the probe for the target. In general, wash conditions are selected tobe approximately 12 20° C. below the Tm of the hybrid. In regards to thenucleic acids of the current invention, a moderate stringencyhybridization is defined as hybridization in 6× SSC, 5× Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA at 42° C.,and washed in 2× SSC and 0.5% SDS at 55° C. for 15 minutes. A highstringency hybridization is defined as hybridization in 6× SSC, 5×Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNAat 42° C., and washed in 1× SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC, 5× Denhardt's solution, 0.5% SDS and 100 μg/ml denatured salmonsperm DNA at 42° C., and washed in 0.1× SSC and 0.5% SDS at 65° C. for15 minutes.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof, that binds to a specific antigen. Theterm includes polyclonal, monoclonal, chimeric, single domain (Dab) andbispecific antibodies. As used herein, antibody or antibody moleculecontemplates recombinantly generated intact immunoglobulin molecules andmolecules comprising immunologically active portions of animmunoglobulin molecule such as, without limitation: Fab, Fab′, F(ab′)2,F(v), scFv, scFv2, scFv-Fc, minibody, diabody, tetrabody, and singlevariable domain (e.g., variable heavy domain, variable light domain).

With respect to antibodies, the term “immunologically specific” refersto antibodies that bind to one or more epitopes of a protein or compoundof interest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicbiological molecules.

The term “isolated” may refer to a compound or complex that has beensufficiently separated from other compounds with which it wouldnaturally be associated. “Isolated” is not meant to exclude artificialor synthetic mixtures with other compounds or materials, or the presenceof impurities that do not interfere with fundamental activity or ensuingassays, and that may be present, for example, due to incompletepurification, or the addition of stabilizers.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionfor performing a method of the invention.

The phrase “solid support” refers to any solid surface including,without limitation, any chip (for example, silica-based, glass, or goldchip), glass slide, membrane, plate, bead, solid particle (for example,agarose, sepharose, polystyrene or magnetic bead), column (or columnmaterial), test tube, or microtiter dish.

As used herein, the term “microarray” refers to an ordered arrangementof hybridizable array elements. The array elements are arranged so thatthere are at least one or more different array elements on a solidsupport. Preferably, the array elements comprise oligonucleotide probes.

As used herein, the term “small molecule” refers to a substance orcompound that has a relatively low molecular weight (e.g., less than2,000). Typically, small molecules are organic, but are not proteins,polypeptides, or nucleic acids.

Chemotherapeutic agents are compounds that exhibit anticancer activityand/or are detrimental to a cell (e.g., a toxin). Suitablechemotherapeutic agents include, but are not limited to: toxins (e.g.,saporin, ricin, abrin, ethidium bromide, diptheria toxin, andPseudomonas exotoxin); taxanes; alkylating agents (e.g., temozolomide,nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide,mechlorethamine, melphalan, and uracil mustard; aziridines such asthiotepa; methanesulphonate esters such as busulfan; nitroso ureas suchas carmustine, lomustine, and streptozocin; platinum complexes (e.g.,cisplatin, carboplatin, tetraplatin, ormaplatin, thioplatin,satraplatin, nedaplatin, oxaliplatin, heptaplatin, iproplatin,transplatin, and lobaplatin); bioreductive alkylators such as mitomycin,procarbazine, dacarbazine and altretamine); DNA strand-breakage agents(e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine,menogaril, amonafide, dactinomycin, daunorubicin, N,N-dibenzyldaunomycin, ellipticine, daunomycin, pyrazoloacridine, idarubicin,mitoxantrone, m-AMSA, bisantrene, doxorubicin (adriamycin),deoxydoxorubicin, etoposide (VP-16), etoposide phosphate, oxanthrazole,rubidazone, epirubicin, bleomycin, and teniposide); DNA minor groovebinding agents (e.g., plicamydin); antimetabolites (e.g., folateantagonists such as methotrexate and trimetrexate); pyrimidineantagonists such as fluorouracil, fluorodeoxyuridine, CB3717,azacitidine, cytarabine, and floxuridine; purine antagonists such asmercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase;and ribonucleotide reductase inhibitors such as hydroxyurea);anthracyclines; and tubulin interactive agents (e.g., vincristine,vinblastine, and paclitaxel (Taxol®)).

Radiation therapy refers to the use of high-energy radiation fromx-rays, gamma rays, neutrons, protons and other sources to target cancercells. Radiation may be administered externally or it may beadministered using radioactive material given internally. Chemoradiationtherapy combines chemotherapy and radiation therapy.

The following examples are provided to illustrate certain embodiments ofthe invention. They are not intended to limit the invention in any way.

EXAMPLE 1 Methods SUMMARY

Twenty probands with neuroblastoma and a family history of the diseasewere identified for study. Eight pedigrees had 3 or more affectedindividuals; six pedigrees contained only two affected individuals, butof first degree relation; and six pedigrees consisted of only twoaffected individuals, but of second, third, or >fourth degreerelationship. A total of 176 individuals (49 affected withneuroblastoma) were genotyped genome-wide, and two families wereexcluded due to insufficient DNA for genotyping. Marker data wassimulated under a model of genetic homogeneity and autosomal dominantinheritance, and the data was analyzed using an affected-only approachcomparable to the model-free approach used in the actual linkageanalysis. Genotype data were checked for Mendelian inconsistencies usingPEDSTATS (Wigginton et al. (2005) Bioinformatics, 21:3445-7), andanalyzed for linkage using MERLIN (Abecasis et al. (2002) Nat. Genet.,30:97-101) and LAMP (Li et al. (2005) Am. J. Hum. Genet., 76:934-49).Regional candidates were re-sequenced using Sanger methodology.Predictions on the probability that DNA sequence alterations encode amutant protein were performed using a support vector machine-basedstatistical classifier (Torkamani et al., (2007) Bioinformatics,23:2918-25; Torkamani et al. (2008) Cancer Res., 68:1675-82).Four-hundred-and-ninety-one primary tumor samples, and 27 cell lineswere used for whole genome SNP-array analyses (550K) to determine copynumber alterations (Maris et al. (2008) N. Engl. J. Med., 358:2585-93).mRNA knockdown of ALK and control targets was achieved with siRNAsagainst each target. siRNA knockdown effects on substrate adherentgrowth was quantified with the RT-CESTm microelectronic cell sensorsystem (ACEA, San Diego, Calif.) (Yu et al. (2006) Anal. Chem.,78:35-43; Cole et al. (2008) Mol. Cancer Res., 6:735-42). Whole celllysates were collected from the cell lines and from siALK and siNTC (nontargeting control) treated cells after transfection. Proteins wereseparated by SDS PAGE gels and immunoblotted using ALK and Phospho-ALKantibodies.

Research Subjects and Samples

Families with a history of neuroblastoma in at least one other relativewere eligible to participate. Only germline DNA from the neuroblastomapedigrees was studied, as no tumor tissue was available. Sporadicneuroblastoma tumor samples with matched constitutional DNA wereacquired from the Children's Oncology Group Neuroblastoma Tumor Bank.The Children's Hospital of Philadelphia Institutional Review Boardapproved this research.

Linkage Analysis

A genome-wide linkage scan was done using the Illumina Linkage IVb SNPpanel. Genotype data were checked for Mendelian inconsistencies usingPEDSTATS (Wigginton et al. (2005) Bioinformatics, 21:3445-7), andanalyzed for linkage using MERLIN (Abecasis et al. (2002) Nat. Genet.,30:97-101) and LAMP (Li et al. (2005) Am. J. Hum. Genet., 76:934-49).The genome-wide screen for linkage was performed with both maximumlikelihood allele frequency estimates and model-free analyses. Since thepattern of inheritance is complex, a model-free approach was used so asnot to assume any specific mode of inheritance. Model-based analyseswere performed for all SNPs included in the critical region under adominant mode of inheritance, assuming four different gene frequencies(0.0001, 0.001, 0.01, and 0.1) and dominant transmission with varyingpenetrance of the disease across a broad range, from 0.0001 to 0.68.Model-based linkage analysis was also performed using the methodimplemented in LAMP, assuming a prevalence of the disease of 0.000143(1/7000), and maximizing the lod-scores over all possible disease models(MOD score analysis). In every analysis, the critical interval wasdefined as the region with associated lod-scores greater than themaximum lod-score minus 3. Since linkage disequilibrium (LD) amongmarkers is known to inflate the lod-scores from linkage analysis in thepresence of missing founders, its impact on the lod-scores was assessedat the chromosome 2p critical interval. To model LD, markers wereorganized into clusters by means of Merlin, which uses populationhaplotype frequencies derived from the HapMap project (www.hapmap.org/).

DNA Sequencing

Shotgun resequencing from templates generated by long PCR for an 18kilobase region surrounding the MYCN locus was performed using a 454Life Sciences instrument (Branford, Conn.) after bi-directionalsequencing of the three coding exons showed no disease causal sequencevariations in the pedigrees. Bi-directional sequencing of ALK codingsequence was performed in the following distinct sample sets: 1)constitutional DNA from the proband and an unaffected first degreerelative from the twenty neuroblastoma pedigrees, with repeat sequencingof amplicons containing any DNA sequence variations and sequencing ofamplicons containing confirmed variations in remaining family members;2) 27 human neuroblastoma-derived cell line DNAs maintained at theChildren's Hospital of Philadelphia; 3) tumor DNA from 167 sporadicneuroblastomas from the Children's Oncology Group tumor bank; and 4) 109normal constitutional DNAs from the SNP500Cancer Resource panelpurchased from the Coriell Institute for Medical Research (Camden, NJ).In order to verify neuroblastoma cell line integrity, all lines wereroutinely genotyped (AmpFLSTR Identifiler kit; Applied Biosystems,Foster City, Calif.), and mycoplasma tested.

Mutation Prediction

Cancer mutant predictions and analysis were performed as described(Torkamani et al. (2008) Cancer Res., 68:1675-82). Briefly, a supportvector machine was trained upon common SNPs (presumed neutral) andcongenital disease causing

SNPs characterized by a variety of sequence, structural, andphylogenetic parameters. Training and predictions were performed usingsomatic mutations occurring within and outside of the kinase catalyticcore separately. The support vector machine-based method was thenapplied to the ALK mutants, and the probability that each mutant is adriver was computed via the support vector machine. The threshold takenfor calling a SNP a driver was taken to be 0.49 for catalytic domainmutations, and 0.53 for all other mutations (Torkamani et al. (2007)Bioinformatics, 23:2918-25). For comparison to previously observedcancer mutations, ALK mutants were mapped to positions of a catalyticcore alignment generated with characteristic site motifs, and previouslyobserved cancer mutants mapping to the same positions were noted(Torkamani et al. (2008) Cancer Res., 68:1675-82).

Tumor Copy Number Analysis

Tumor samples were assayed on the Illumina Infinium™ II HumanHap550BeadChip technology (Illumina, San Diego, Calif.), at the Center forApplied Genomics at the Children's Hospital of Philadelphia. A total of750 nanograms of genomic DNA was used as input in each case, and theassay was performed and data analyzed following the manufacturersrecommendations and as previously described (Maris et al. (2008) N.Engl. J. Med., 358:2585-93).

Quantitative mRNA Expression

Relative ALK expression was determined using the 2-ΔΔCt method (Livak etal. (2001) Methods 25:402-8), using GAPDH as the endogenous control andusing the second dCT as the lowest expressed cell line, using methods aspreviously described (Cole et al. (2008) Mol. Cancer Res., 6:735-42).

ALK siRNA Knockdown in Neuroblastoma Cell Lines

A total of 1-5×10⁴ neuroblastoma cells were plated in triplicateovernight in antibiotic-free complete media in the 96 well RT-CESTMmicroelectronic cell sensor system (ACEA, San Diego, Calif.) (Yu et al.(2006) Anal. Chem., 78:35-43; Cole et al. (2008) Mol. Cancer Res.,6:735-42). The cells were then transiently transfected with 200 μlcontaining 50 nM of pooled siRNAs (four separate siRNAs per transcripttargeted) against ALK (catalog #M-003103-02), GAPDH (catalog#D-001140-01-20) negative control, non targeting negative control, orPLK1 (catalog #M-003290-01) positive control (siGENOME SMARTpool siRNA,Dharmacon, Lafayette, Colo.) using 0.1% v/v Dharmafect I, according tothe manufacturer's protocol (Dharmacon, Lafayette, Colo.). The fourseparate ALK-directed siRNA sense direction sequences are:

ALK J-003103-10 GGGCCUGUAUACCGGAUAAUU (SEQ ID NO: 1) ALK J-003103-11GUGCCAUGCUGCCAGUUAAUU (SEQ ID NO: 2) ALK J-003103-12CCGCUUUGCCGAUAGAAUAUU (SEQ ID NO: 3) ALK J-003103-13GGAGCCACCUACGUAUUUAUU (SEQ ID NO: 4)In brief, 35 μL of 1 μM siRNA and 35 μL of serum-free media werecombined with 0.7 μL Dharmafect I in 70 μL of serum-free media andincubated for 20 minutes at room temperature, and then 560 μL ofantibiotic-free complete media was added. The culture media was gentlyremoved from the plated cells and replaced by 200 μL of fresh mediacontaining the siRNA, mock or complete media. Cell growth was monitoredcontinuously and recorded as a cell index (CI) every 30 minutes for aminimum of 96 hours. The “Cell Index” (CI) is derived from the change inelectrical impedance as the living cells interact with the biocompatiblemicroelectrode surface in the microplate well effectively measuring cellnumber, shape and adherence. Forty-eight hours after siRNA transfection,total RNA was extracted from the cells that had been plated in aparallel 96-well plate using the Qiagen (Valencia, Calif.) miniextraction kit, with DNAase treatment. Two hundred ng of total RNA wasoligodT primed and reverse transcribed using Superscript II reversetranscriptase (Invitrogen, Carlsbad, Calif.). ALK, HPRT, GAPDH and PPIBexpression levels were measured by quantitative RT-PCR using Taqmanggene expression assays (ABI, Foster City, Calif.), quantified oncorresponding standard curves and normalized to the geometric mean ofthe three housekeeping genes. Two independent experiments were performedin triplicate. Growth inhibition of the neuroblastoma cell lines wasdetermined by comparing the siRNA against ALK growth curve to thatagainst GAPDH at the time of maximum cell index (CI_(max)): % growthinhibition=(1-C_(siALK)/CI_(siGAPDH))×100. ALK and GAPDH knockdown wasdetermined by comparing the relative ALK expression: %knockdown=(1-ALK_(siALK)/ALK_(sicoNTRoL))×100. The average % knockdownof ALK across all cell lines was 60% (range 21%-86%). The average %knockdown of GAPDH was 75% (range 61%-95%).

ALK Protein and Phosphoprotein Detection

Neuroblastoma cell lines were grown in T75 flasks under standard cellculturing conditions. For KELLY and SKNDZ lysates collected from thesiRNA knockdown experiments, cells were plated in T25s, transfected with10 nM siRNA (as above) and collected at 24, 48 and 72 hours aftertransfection. At 60-80% confluency (or the appropriate time point), thecells were collected, pelleted and washed twice with ice cold PBS. Wholecell lysates were extracted with 100 uL Cell Extraction buffer(Invitrogen FNN011) containing protease inhibitors (Sigma, P-2714) andPMSF, briefly sonicated and rotated for 1 hour at 4° C. After a 30minute centrifugation at 4° C., the supernatant was removed and proteinquantification was performed using the Bradford method. Lysates (50 ugfor siRNA experiment and 100 ug for native cell lines) were separated on4-12% Bis-Tris gradient gels and transferred to PVDF membranes.Membranes were then incubated and washed according to the Cell SignalingWestern protocol with 1:1000 ALK (Cell Signalling, #3333) andPhospho-ALK (Cell Signalling, #3341) and 1:5000 actin (Santa Cruz,sc-2352).

Results Identification of Germline ALK Mutations

To identify the location of a hereditary neuroblastoma predispositiongene, a genome-wide scan for linkage at ˜6000 single nucleotidepolymorphisms (SNPs) in 20 neuroblastoma families was performed. Becauseof the rarity of the condition, the genome-wide scan included pedigreeswith varying degrees of confidence of actual heritability. Eightfamilies had three or more affected individuals of close relation (highconfidence), whereas 6 families consisted of only two individuals offirst-degree relation (moderate confidence), and 6 families also onlyconsisted of two affected individuals, but of more distant relationship(low confidence). A significant linkage signal at chromosome 2p wasdiscovered with a maximum nonparametric LOD score of 4.23 at rs1344063in 18 of the families (two excluded due to insufficient DNA).

This refined a region previously reported for one of the pedigreesstudied here (Longo et al. (2007) Hum. Hered., 63:205-11). By mappinginformative recombination events, a predisposition locus was defined atchromosome bands 2p23-p24 delimited by SNPs rs1862110 and rs2008535 with104 genes including the known neuroblastoma oncogene, MYCN (Schwab etal. (1984) Nature, 308:288-91; Weiss, et al. (1997) EMBO J.,16:2985-2995), and the ALK oncogene located 13.2 Mb centromeric. Despiteprevious work showing that forced overexpression of MYCN to the murineneural crest causes neuroblastoma (Weiss, et al. (1997) EMBO J.,16:2985-2995), resequencing of the MYCN coding region and 18 Kb ofsurrounding genomic DNA in probands from each linked family showed nodisease-causal sequence variations.

The anaplastic lymphoma kinase gene (ALK) was subsequently studiedbecause ALK had been previously identified as a potential oncogene inneuroblastoma through somatically acquired amplification of the genomiclocus (Osajima-Hakomori et al. (2005) Am. J. Pathol., 167:213-22; Georgeet al. (2007) PLoS ONE 2:e255). In addition, oncogenic fusion proteinsleading to constitutive activation of the ALK kinase domain occur inmany human cancers including anaplastic large cell lymphoma (Morris etal. (1994) Science, 263:1281-4), inflammatory myofibroblastic tumors(Griffin et al. (1999) Cancer Res., 59:2776-80), squamous cellcarcinomas (Jazii et al. (2006) World J. Gastroenterol., 12:7104-12),and non-small cell lung cancers (Soda et al. (2007) Nature, 448:561-6;Rikova et al. (2007) Cell, 131:1190-203). ALK is a single chain receptortyrosine kinase and a member of the insulin receptor superfamily.Expression is normally detected in developing central and peripheralnervous system. Further, ALK is an orphan receptor as its ligand is notknown. The normal function of ALK is also not known. Indeed, a murineknockout shows no phenotype. Mutations within ALK have not beenpreviously described as mechanism of oncogenicity.

Resequencing of the 29 ALK coding exons identified three separate singlebase substitutions within the ALK tyrosine kinase domain in eight of theprobands screened (FIG. 1, Table 1). These DNA sequence alterations werenot present in single nucleotide polymorphism (dbSNP;www.ncbi.nlm.nih.gov/projects/SNP/) or somatic mutation (COSMIC;www.sanger.ac.uk/genetics/CGP/cosmic/) databases, and were not detectedin direct sequencing of the ALK tyrosine kinase domain in 218 normalcontrol alleles. Each substitution was subsequently shown to segregatewith the disease within each family (FIG. 1). The sequence variation inFNB12 (R1275Q) appears to have been acquired de novo in the affectedfather, and non-paternity was excluded by analysis of inheritance ofgenotypes within this pedigree. There are several asymptomatic obligatecarriers identified (FNB2, FNB13, FNB32, FNB52, FNB56), suggesting thatthe incomplete penetrance of this disease may be due to lack of theacquisition of a second hit, or alternatively spontaneous regressionfollowing malignant transformation in at least a subset of cases.Notable is the very large multiplex family (FNB52) with discordance intwins and multiple unaffected carriers that segregates a unique germlinemutation (G1128A).

TABLE 1 ALK mutations in neuroblastoma. Probability cDNA ActivatingMutation Variation Type/Frequency Region¹ Mutation² G1128A c.3383G > CGermline (1/8) P-Loop 0.95 R1192P c.3575G > C Germline (2/8) β4 Strand0.96 R1275Q c.3824G > A Germline (5/8) Activation 0.91 Somatic (8/24)Loop D1091N c.3271G > A Somatic (1/24) N- 0.29 Terminal M1166R c.3497T >G Somatic (1/24) C-Helix 0.79 I1171N c.3512T > A Somatic (2/24) C-Helix0.85 F1174I c.3520T > A Somatic (1/24) End of 0.92 C-Helix F1174Lc.3522C > A Somatic (8/24) End of 0.96 C-Helix F1245C c.3734T > GSomatic (1/24) Catalytic 0.94 Loop F1245V c.3733T > G Somatic (1/24)Catalytic 0.91 Loop I1250T c.3749T > C Somatic (1/24) Catalytic 0.87Loop ¹The region in which the codon alteration occurs is indicated. Notethat the D1091N is immediately adjacent to the tyrosine kinase domain.²The probability that the amino acid alteration results in oncogenicactivation based on the methods of Torkamani and Schork (Torkamani etal. (2007) Bioinformatics, 23: 2918-25).

An exemplary full-length ALK sequence is provided in GenBank AccessionNo. NM_(—)004304.3 (FIG. 9), although variants (e.g., natural allelicvariants) of the ALK sequence are also encompassed by the instantinvention.

ALK sequence variations occurred only in the families with high ormoderate degrees of confidence for harboring a predisposing allele. Sixof the eight families with three or more affected individuals had ALKmissense alterations. The two families that did not have ALK sequencealterations identified were each shown to harbor mutations in thesympathicoadrenal lineage specific PHOX2B neurodevelopmental gene (Mosseet al. (2004) Am. J. Hum. Genet., 75:727-30; Raabe et al. (2008)Oncogene 27:469-76). Two of the six families consisting of only twoaffected individuals, but of first-degree relation, had ALK sequencevariations. Each of these families carried the R1275Q alteration, and inFNB12 it is shown that the mutation arose de novo in the affectedfather, whereas in FNB56 the alteration was inherited from an unaffectedfather (FIG. 1). None of the six families with two distant relationsaffected with neuroblastoma showed ALK alterations, suggesting that theoccurrence of an additional case of this relatively rare disease in anextended family member was likely a chance occurrence. Since there areseveral families who share identical mutations, it was determined ifthese families shared a common haplotype around the ALK gene and showedthat the affected individuals with the same mutations did not sharehaplotypes, arguing against a founder effect.

Because ALK functions as an oncogene in other human cancers, it waspredicted that the sequence variations discovered in the neuroblastomapedigrees would result in constitutive activation. Therefore, a supportvector machine-based statistical classifier was used to map the putativemutations and determine the probability that they would act as driversof an oncogenic process (Torkamani et al. (2007) Bioinformatics,23:2918-25; Torkamani et al. (2008) Cancer Res., 68:1675-82). Each ofthe germline alterations occurred at regions of the ALK kinase domainthat have been shown to be major targets for cancer driver mutations inother oncogenic kinases (Table 1, FIG. 2). The R1275Q mutation waspresent in the germline DNA of affected individuals from five pedigrees(FIG. 1), and falls within the kinase activation loop in a regionstrongly associated with activating mutations in many different proteinkinases, such as BRAF (Ikenoue et al. (2003) Cancer Res., 63:8132-7).This amino acid substitution results in an electropositive residue beingreplaced by a more electronegative one, possibly mimicking activatingphosphorylation events. The R1192P mutation occurred at the beginning ofthe f34 strand of the kinase domain, and although it is predicted to bea driver mutation with high confidence (Table 1) the mechanism foractivation is not yet clear (Torkamani et al. (2008) Cancer Res.,68:1675-82). The G1128A was seen only in the large pedigree withaffected individuals in a single generation. The variation falls at thethird glycine of the glycine loop, and identical mutations of thisglycine to alanine in BRAF have been shown to increase kinase activity(Ikenoue et al. (2004) Cancer Res., 64:3428-35).

Identification of Somatic ALK Mutations

Having shown that heritable mutations in the ALK tyrosine kinase domainare associated with a highly penetrant predisposition to developneuroblastoma, it was determined if ALK activation might also besomatically acquired. A representative set of 491 sporadically occurringprimary neuroblastoma samples acquired from children at the time ofdiagnosis was examined on a 550K SNP-based microarray to assess forgenome-wide copy number alterations. A total of 112 cases (22.8%) showedunbalanced gain of a large genomic region at 2p including the ALK locus(partial trisomy), and an additional 16 cases (3.3%) showed high-levelfocal amplification of ALK (FIG. 3). Each of the high-levelamplifications co-occurred with MYCN amplification and/or other regionsat 2p, except one case with an ALK amplicon only. The presence ofaberrant ALK copy number status (gain or amplification) was highlyassociated with an aggressive clinical phenotype such as metastasis atdiagnosis (P<0.0001) and death from disease (P=0.0003).

Because of the association of ALK gain and amplification with high-riskdisease features, a subset of 167 tumor samples from high-risk patients,and 27 human neuroblastoma-derived cell lines (all from high-riskpatients), was examined for sequence alterations in the ALK tyrosinekinase domain. Fourteen of the 167 tumor (8.4%) and 10 out of 27 cellline (35.7%) samples showed single base substitutions consistent withactivating mutations (FIG. 2). Eight separate single base substitutionswere identified, with the R1275Q mutation being the only mutation alsoseen in the germline DNA of the families studied. Again, none of thesequence variations discovered here were in SNP databases or wereidentified in the resequencing of the ALK tyrosine kinase domain in 109control subjects (218 alleles).

Mutations were equally distributed between cases with and without MYCNamplification. Only one case had a co-occurrence of an ALK mutation (F1174L) and genomic amplification of the ALK locus, and in this case themutated allele was amplified. Germline DNA was available for 9/14patients with ALK mutations, and in one of these cases the sequencealteration (I1250T) was also present in the germline suggesting ahereditary predisposition that may or may not be de novo in this case.

Using the same statistical classifier employed for the germlinemutations, it was shown that all but one of the sequence variationsdiscovered in the tumor tissues were predicted to be activatingmutations (Table 1), and the one that shows a low probability (D1091N)was outside of the core kinase domain. The vast majority of thesomatically acquired mutations fell into either the catalytic loop orC-helix kinase domains, both frequent sites for oncogenic activatingmutations (FIG. 2). Catalytic loop mutants, especially I1250T, maypromote oncogenesis by altering substrate binding or, more likely, alterpacking of the HRD and DFG motifs towards an activated conformation(Kannan et al. (2005) J. Mol. Biol., 351:956-72). The mutations observedin the ALK C-helix domain occurred at positions within the kinase domainpreviously observed to be mutated in other tumors. I1171N falls at anequivalent weakly oncogenic position in MET (M1149T) (Jeffers et al.(1997) Proc. Natl. Acad. Sci., 94:11445-50), and the M1166R, F11741 andF1174L mutants fall at equivalent positions mutated in ErbB2 (D769,V777) and EGFR (D761, V769) (Balak et al. (2006) Clin. Cancer Res.,12:6494-501; Lee et al. (2006) Cancer Lett., 237:89-94; Lee et al.(2006) Clin. Cancer Res., 12:57-61).

Functional Consequences of ALK Mutations

Various genes are differentially expressed in human primaryneuroblastomas, with higher expression sometimes seen in the mostaggressive subset of tumors (Wang et al. (2006) Cancer Res.,66:6050-62). It is shown herein ALK is highly expressed in all but oneof 20 human neuroblastoma-derived cell lines using quantitative RT-PCR.ALK expression was higher in neuroblastoma cells compared to developingfetal brain, and cell lines harboring ALK mutations (N=6) expressed themRNA at significantly higher copy number than ALK wild-type cell lines(N=14, FIG. 4A). Analysis of protein lysates from a panel ofneuroblastoma cell lines showed constitutive phosphorylation of thetyrosine residue at codon 1604 in each of the cell lines harboringmutations, with weak phosphostaining in two wild-type cell lines (FIG.4B).

To determine if ALK activation via mutation and/or amplification isfunctionally relevant in models of high-risk neuroblastoma, and thus bea tractable therapeutic target, the consequences of disrupting ALKsignaling via knockdown of messenger RNA was examined. siRNAs directedagainst ALK (Dharmacon, Lafayette, Colo.) were transiently transfectedinto 10 neuroblastoma cell lines and screened for inhibition ofsubstrate adherent growth. The knockdown of the mRNA and protein wasdemonstrated in all lines studied, but showed differential effect oncellular proliferation (FIGS. 5A-5L). Each of the cells harboring ALKmutation or amplification showed profound inhibition of proliferation toALK knockdown. In addition, 2/6 of the ALK wild-type cell lines showedsignificant inhibition of growth with ALK knockdown and each of thesehad shown weak evidence for phosphorylation at tyrosine 1604 (FIG. 4 b),suggesting an alternative mechanism may have resulted in ALK kinaseactivation in these two cell lines.

Currently, the frequency of ALK alterations in neuroblastoma are:mutations in the TK domain: ˜10%, mutations in the extracellular domain:˜5%, and amplification: ˜8%. p-ALK is detectable in 20/134 (15%) of NBLtissue samples.

EXAMPLE 2

ALK is a tractable target for pharmacologic inhibition, but sensitivitydepends on mutation type. FIG. 6A is a graph of a dose response curveand FIG. 6B is a graph of the % growth inhibition with PF066 at 333 nM.FIG. 7 shows the expression of pALK, pAKT, pSTAT3, and pMAPK3. For FIG.6A, the human-derived neuroblastoma cell lines NB1643 (R1275Q), NB1 (ALKamplified), and NBSD (Fl 174L) were screened for evidence of anti-tumoractivity to ALK inhibitor PF-02341066 in vitro. A quantitative assay toevaluate growth inhibition in a multi-well was used in a parallel formatto screen for cellular cytotoxicity. Inhibition of substrate adherentgrowth during log-phase was then screened using the 96×6 RT-CES™ system(Real-Time Cell Electronic Sensing; ACEA Biosciences; San Diego, Calif.)with cells plated in triplicate for each assay, allowing for relativelyhigh throughput and real-time assessment of alterations in growthkinetics, assaying for potential cytostatic or cytotoxic responses. Thecompound was studied at a minimum of 10 dose levels to determine theIC₅₀ values by concentration-response curves across a 4-log dose range.For FIG. 6B, the proliferation of neuroblastoma cell lines was measuredafter 72 hours of incubation with PF234I066 (333 nM) in DMSO using theRT-CES™ system. Cell lines displayed differential sensitivity dependingon ALK status (p=0.01). Cell lines with ALK mutations and one cell linewith amplification of wild type (WT) ALK were sensitive. R1275Qmutations were more sensitive than F1174L mutations. No cell lines withnormal copy number WT ALK showed significant inhibition. Inhibition ofgrowth %=100* (cell index vehicle—cell index treatment)/cell indexcontrol. For FIG. 7, the biochemical consequences of ALK activation anddownstream signaling pathways were studied in ALK-mutant lines. Theseexperiments quantify native and phosphorylated ALK, STAT3 and MAPK3 inALK-mutated cell lines treated with PF-02341066.

FIG. 8A provides the IC₅₀ of various drugs on the neuroblastoma cellline KELLY (F 1147L). FIG. 8B provides graphs of tumor volume afterweeks of administration of PF066. For FIG. 8A, the human-derived NB cellline KELLY was screened for evidence of anti-tumor activity to selectiveALK inhibitors in vitro. A quantitative assay was used to evaluategrowth inhibition in a multi-well parallel format to screen for cellularcytotoxicity. Inhibition of substrate adherent growth during log-phasewas screened for using the 96×6 RT-CESTm system with cells plated intriplicate for each assay, allowing for relatively high throughput andreal-time assessment of alterations in growth kinetics, assaying forpotential cytostatic or cytotoxic responses. Each available compound wasstudied at a minimum of 10 dose levels to determine the IC₅₀ values byconcentration-response curves across a 4-log dose range. For FIG. 8B, anintervention design and initiate therapy was used when tumors in miceare palpable at 200 mm³ as the starting volume. A total of 20 mice wererandomized to treatment with an ALK inhibitor or vehicle, and serialmeasurements were performed using an electronic caliper system. Tumorvolume is expressed as the mean tumor volume +/− standard error forgroups of mice and tumor growth kinetics over time and progression freesurvival were compared. ALK neuroblastoma mutants can be mapped onto thecMet-PF'1066 crystal structure. ALK R1275Q maps to c-Met R1227 which isnot expected to destabilize the conformation of the activation loopresidues important for binding PF-1066. ALK F1174L maps to c-Met F1124:This phenylalanine is highly conserved in ALK and sits in a hydrophobicpocket necessary for correctly positioning/stabilizing the conformationof residues 1222-1228. Mutation of this residue is expected tosignificantly decrease PF-1066 binding. Accordingly, this modeling isconsistent with the data provided in FIG. 8B.

EXAMPLE 3

Neuroblastoma is a cancer of early childhood that arises from thedeveloping autonomic nervous system. It is the most common malignancydiagnosed in the first year of life and shows a wide range of clinicalphenotypes with some patients having tumors that regress spontaneously(D′Angio et al. (1971) Lancet 1:1046-1049), whereas the majority ofpatients have aggressive metastatic disease (Maris et al. (2007) Lancet369:2106-2120). Neuroblastoma remains an important clinical problem asit continues to be a leading cause of childhood cancer mortality despitedramatic escalations in dose-intensive chemoradiotherapy, and long-termsurvivors experience significant treatment related morbidity (Oeffingeret al. (2006) N. Engl. J. Med., 355:1572-1582; Hobbie et al. (2008)Pediatr. Blood Cancer 51:679-683). To improve outcome and makeparadigm-shifting advances in this disease, it is necessary to discoverthe key oncogenic drivers of the malignant process and exploit thesetherapeutically.

The anaplastic lymphoma kinase (ALK) oncogene is a receptor tyrosinekinase first identified after recurrent t(2;5) translocations inanaplastic large cell lymphoma (ALCL) were shown to fuse the aminoterminus of nucleophosmin to a previously unidentified gene at 2p23(Morris et al. (1994) Science 263:1281-1284). The ALK gene encodes a1620-amino acid protein that undergoes post-translational N-linkedglycosylation, and expression is restricted to the developing centraland peripheral nervous system with a postulated role in regulation ofneuronal differentiation (Iwahara et al. (1997) Oncogene 14:439-449). Ithas recently become clear that other human cancers in addition to ALCLactivate ALK signaling through unique oncogenic fusions of the ALK genewith a variety of partners, including inflammatory myofibroblastictumors (Griffin et al. (1999) Cancer Res., 59:2776-2780), squamous cellcarcinomas (Jazii et al. (2006) World .1. Gastroenterol., 12:7104-7112)and non-small-cell lung cancers (Soda et al. (2007) Nature 448:561-566;Rikova et al. (2007) Cell 131:1190-1203). A recent phase 1 trial of thedual MET/ALK kinase inhibitor PF-02341066 showed safety and significantanti-tumor activity in patients with refractory solid tumors harboringan ALK translocation (Kwak et al. (2009) J. Clin. Oncol., 27:15s (abstr3509)).

Until recently, somatically acquired chromosomal translocation eventswere the only known mechanism for constitutively activating the ALKkinase. As explained herein, an unbiased linkage screen in familialneuroblastoma was performed and activating mutations in the tyrosinekinase domain of ALK were identified as the major cause of hereditarydisease. Additionally, somatically acquired genomic amplification of ALKand mutations in the kinase domain as presumed oncogenic drivers insporadic (nonfamilial) disease were also identified (see herein as wellas Caren et al. (2008) Biochem. J., 416:153-9; Chen et al. (2008) Nature455:971-974; George et al. (2008) Nature 455:975-978; Janoueix-Leroseyet al. (2008) Nature 455:967-970). As explained herein, early dataobtained with the discovery of oncogenic mutations in this geneindicated that siRNA and pharmacologic inhibition of ALK signaling incells harboring a mutation resulted in cytotoxicity, further indicatingthat ALK mutation/amplification acts as a dominant oncogenic driver. Keyto impacting patient outcome with ALK-directed therapy was first todefine the cohort of subjects who are most likely to benefit from ALKinhibition, and thus the prior mutation screen restricted to the“high-risk” subset was extended to cover all neuroblastoma phenotypicsubsets. Second, preclinical experiments were performed to prove thatthe lead pharmacologic ALK-inhibitor showed anti-tumor activity directlydue to on-target efficacy. The data presented here led directly to anongoing pediatric Phase 1/2 trial of the PF-02341066 inhibitor that hasshown safety and activity in ALK-activated adult solid malignancies.

Methods Research Subjects and Samples

Neuroblastoma tumor samples were acquired from the Children's OncologyGroup Neuroblastoma Tumor Bank. The Children's Hospital of PhiladelphiaInstitutional Review Board approved this research.

DNA Sequencing

Bidirectional sequencing of the ALK coding sequence was performed byAgencourt Biosciences on tumor DNA from 593 sporadic neuroblastomas fromthe Children's Oncology Group (COG) tumor bank.

Tumor Copy Number Analysis

Tumor DNA from 591 sporadic neuroblastomas from the Children's OncologyGroup (COG) tumor bank were assayed on the Illumina Infinium™ IIHumanHap550 BeadChip as described above. DNA copy number for eachindividual tumor was estimated using OverUnder (Attiyeh et al. (2009)Genome Res., 19:276-83). Samples with a relative DNA copy number≧4.5over the entire ALK locus were considered amplified, whereas sampleswith a relative DNA copy number≧2.3 were defined as having low-levelgain of ALK. Tumors harboring low-level gain across≧90% of chromosome 2were defined as whole chromosome (WC) gains; all other gains wereconsidered regional. To assess the statistical significance of recurrentregional gain/amplification on chromosome 2, the STAC algorithm (Diskinet al. (2006) Genome Res., 16:1149-1158) was applied using 1,000 randompermutations of the regional gains identified in 591 primary tumors.Both the frequency and footprint statistics in STAC were evaluated, andan adjusted p-value≦0.05 was considered statistically significant.Tumor Quantitative mRNA ExpressionTumor RNA from 96 sporadic neuroblastomas from the Children's OncologyGroup (COG) tumor bank was assayed on the Illumina Expression H6 v2.Cell Line Quantitative mRNA ExpressionTotal RNA was isolated from cell lines according to QIAGEN's miRNeasyprotocol. Real-time PCR using TaqMan® Gene Expression Assays wasperformed according to the manufacturer's instructions (AppliedBiosystems). All primer/probe sets spanned exon boundaries to assurespecificity for cDNA. Relative expression of anaplastic lymphoma kinase(ALK) was determined by normalization to the geomean of peptidyl-prolylcis-trans isomerase B (PPIB) and hypoxanthine phosphoribosyl-transferase(HPRT I) using a standard curve method. All RTPCR experiments included anon-template control and were done in triplicate.MET siRNA Knockdown in Neuroblastoma Cell LinesThis was performed in 4 cell lines using the 96-well RT-CESmicroelectronic cell sensor system as described above.

DNA Constructs and Retrovirus Production

Four sequence variants were introduced into the full-length ALK cDNAusing site-directed mutagenesis (Origene Technologies, Rockville, Md.).All mutations and overall cDNA integrity were confirmed by sequencing ofthe entire ALK open reading frame. The mutant cDNAs, as well as NPM-ALKand wild-type ALK cDNAs, were cloned into the pCMV-XLS vector andsubcloned into pIRES-EGFP. Infection of retinal pigment epithelial cellsthat express telomerase (HTERT-RPE1) was performed as follows: Phoenix™Ampho cells (Oribigen—RVC-10001) were plated ˜500,000 cells in a 6 wellplate in DMEM media with 10% FBS, 1% Pen/Strep, Gentamicin. Twenty-fourhours after plating (˜50% confluent), Phoenix™ cells were transfected(Eugene) with retroviral vector MigR1 containing the ALK constructsfollowing the Eugene® protocol (using 6:1 dilution of Fugene:plasmidDNA). HTERT-RPEI cells were plated, ˜500,000 cells per well of 6 wellplate, and then harvested virus-containing media 48 hours posttransfection. Media was removed from Phoenix™ cells and filtered through0.45 μm syringe. Growth media on HTERT-RPE1 cells was replaced withvirus cocktail (2 ml growth media, 1 ml filtered viral media, 4 μg/mlPolybrene® (Santa Cruz)) and incubated overnight. Viral media onHTERT-RPE1 cells was replaced on day 5 with fresh growth media andincubated ˜48 hours. HTERT-RPE1 cells then sorted in cell sorter for GFPpositive cells.

In Vitro Tumor Growth Inhibition

In vitro activity of PF-02341066 (Pfizer) dissolved in dimethylsulfoxide (DMSO) was evaluated in 18 neuroblastoma cell lines using theRT-CES system (ACEA Biosciences, San Diego, Calif.) that measureselectrical impedance of adherent cells, providing real-timequantification of cell proliferation. Cell lines were plated at a rangeof 5,000 to 30,000 cells per well depending on growth kinetics and drugwas added 24 hours later across a 4-log dose range (1-10,000 nM) intriplicate. The IC50 was calculated from the cell index after 72 hoursof incubation using a variable slope (Graph pad Prism Version 5.0).Growth inhibition at 333nM PF-02341066 was calculated using the formula:% Inhibition=100*(Cell index control—cell index treatment)/Cell indexcontrol. Due to non-comparable maximum growth inhibition depending onALK status, we analyzed growth inhibition at a single pharmacologicallyrelevant dose. To verify cell line integrity, all lines were routinelygenotyped (AmpFLSTR® Identifiler® kit; Applied Biosystems) andmycoplasma tested.

In Vitro Protein and Phosphoprotein Detection

Each neuroblastoma cell line was cultured in ten T75 flasks understandard cell culture conditions. At 70-80% confluence PF-02341066 wasadded to cell culture medium to achieve a designated final concentrationat one of ten doses ranging from 0 nM to 10,000 nM. Cells were incubatedfor 2 hours with drug, then collected, pelleted, and washed twice withice cold PBS. Whole cell lysates were then harvested, separated andimmunoblotted as described herein. The following antibodies were usedaccording to manufacturers' instructions: anti-ALK (1:1,000; CellSignaling, 3333), anti-phospho-ALK Tyr 1604 (1:1,000, Cell Signaling,3341), anti-STAT3 (1:1,000; Cell Signaling, 9132), antiphospho-STAT3 Tyr705 (1:1,000; Cell Signaling, 9145), anti-AKT (1:1,000; Cell Signaling,9272), anti-phospho-AKT Ser 473 (1:1,000; Invitrogen, 44-621G),anti-p44/42 MAPK (ERK1/2) (1:1,000; Cell Signaling, 4695),anti-phospho-p44/42 MAPK (ERK 1/2) (1:1,000; Cell Signaling, 9101),anti-actin (1:2,000; Santa Cruz, sc-1616).

In Vivo Tumor Growth Inhibition

CB17 scid female mice (Taconic Farms, N.Y.) were used to propagatesubcutaneously implanted neuroblastoma tumors. Tumor diameters weremeasured twice per week using electronic calipers. Tumor volumes werecalculated using the spheroid formula, (π/6)*d³, where d represents themean diameter. Once tumor volume exceeded 200 mm³ mice were randomized(n=10 per arm) to receive PF-02341066 100mg/kg/dose or vehicle(acidified water) daily by oral gavage for four weeks. Mice weremaintained under the protocols and conditions approved by ourinstitutional animal care and use committee. Mice were sacrificed whentumors were greater than 1500 mm₃.

In Vivo Protein and Phosphoprotein Detection

CB17 scid female mice (Taconic Farms, N.Y.) were used to propagatesubcutaneously implanted NB1643 neuroblastoma tumors. Once tumor volumeexceeded 300 mm³ mice were randomized (n=3 per arm) to receivePF-02341066 100mg/kg/dose or vehicle (acidified water) daily by oralgavage for 2 days. Mice were sacrificed 4 hours after the final dose toharvest xenografts, which were immediately snap frozen in liquidnitrogen. Frozen xenografts were pulverized and whole cell lysates wereextracted using 100 μL extraction buffer (FNN011, Invitrogen) containingprotease inhibitor (P-2714, Sigma), phosphatase inhibitors (P-5726,Sigma) and phenylmethyl sulphonyl fluoride. Lysates were sonicated, androtated for 1 hour at 4° C. Following centrifugation at 4° C. for 30minutes, the supernatant was removed, and protein quantification wasperformed using the Bradford method. Lysates (200 μg) were separated on4% -12% Bis-Tris gradient gels and transferred to PVDF membranes whichwere immunoblotted according to manufacturers' instructions: anti-ALK(1:1,000; Cell Signaling, 3333), anti-phospho-ALK Tyr 1604 (1:1,000,Cell Signaling, 3341), and anti-actin (1:5,000; Santa Cruz, sc-2352).

Homology Modeling

The wild-type ALK sequence alignment to c-Met was performed in the PRIMEsuite within Maestro 8.5 (Schrodinger LLC, New York). Using thefull-length ALK sequence and the sequence derived from the crystalstructure of the kinase domain of c-Met with PF-02341066 (PDBentry=2WGJ), the sequences were aligned automatically followed by manualediting of gap areas. The final sequence alignment is shown in FIG. 15,where the F1174 and R1275 mutation sites are highlighted in gray. PRIMEwas then used to build a homology model of wild-type ALK withPF-02341066 bound using the c-Met/PF-02341066 crystal structure (2WGJ)as the template. ALK mutations were modeled as point mutations in theresulting wild-type ALK homology model.

Statistical Analysis

Associations of ALK mutation status and ALK amplification status withaccepted neuroblastoma risk factors were tested using a Fisher's ExactTest (Table 2). Mixed-effects linear model was used to test tumor volumeover time between treatment and vehicle groups controlling for tumorsize at enrollment. The tumor size was transferred by logarithm beforedata analysis. Survival analysis was performed using the Log-Rank testwith progression defined as tumor volume exceeding 1500 mm³ or treatmentrelated death. A P-value of 0.05 or less was considered to indicatestatistical significance. All data analyses were conducted with the useof SAS.

Results ALK Mutations are Restricted to the Tyrosine Kinase Domain andOccur in all Phenotypic Subsets

Comprehensive re-sequencing of all 29 ALK coding exons and 500 basepairs of flanking sequence was performed in 188 high-risk diagnosticprimary neuroblastoma tumors as part of the NBL-TARGET initiative(Neuroblastoma-Therapeutically Applicable Research to Generate EffectiveTreatments; target.cancer.gov/). The prior work presented the resultsfrom 167 of these samples restricted to the kinase domain only and itwas sought to determine if non-kinase domain sequence alterations wereputatively pathogenic. In the extracellular domain, seven nonsynonymoussequence variations were discovered and validated that were not reportedin any of the SNP databases (Table 2). Only one of these was somatic(M770I), whereas five showed the same alteration in the germline DNA(one sample did not have matched germline DNA available). Fivenonsynonymous sequence variations were found in the 3′ untranslatedportion of the gene, but 3/3 with matched germline DNA showed the samesequence variation in the constitutional DNA. Taken together, these dataindicate that somatically acquired sequenced variations outside of theALK tyrosine kinase domain are uncommon in neuroblastoma.

TABLE 2A Nonsynonomous sequence variations within the ALK tyrosinekinase domain (N = 594). Number with DNA sequence Protein codingalteration in variation variation Frequency germline DNA c.3452C > TT1151M 1 (2%) N/A c.3497T > G M1166R 1 (2%) 0/1 c.3509T > G I1170S 1(2%) 0/1 c.3512T > A I1171N 2 (5%) 0/2 c.3521T > G F1174C 1 (2%) 0/1c.3520T > A F1174I 1 (2%) 0/1 c.3522C > A F1174L 7 (16%) 0/4 c.3586C > AL1196M 1 (2%) N/A c.3599C > T A1200V 1 (2%) N/A c.3610C > T L1204F 1(2%) 1/1 c.3734T > G F1245C 2 (5%) 0/1 c.3733T > A F1245I 1 (2%) 0/1c.3733T > G F1245V 2 (5%) 0/2 c.3749T > C I1250T 1 (2%) 1/1 c.3824G > AR1275Q 20 (47%)  0/17

TABLE 2B Nonsynonomous sequence variations outside of the ALK tyrosinekinase domain (N = 167). Number with DNA sequence Protein codingalteration in variation variation Frequency germline DNA c.106C > T P36S1 (1%) 1/1 c.469C > T P157S 1 (1%) N/A c.592G > A V198M 1 (1%) 1/1c.776G > A R259H 1 (1%) 1/1 c.1918G > A G640R 1 (1%) 1/1 c.2310G > TM770I 1 (1%) 0/1 c.2978A > G D993G 1 (1%) 1/1 c.3271G > A D1091N 1 (1%)N/A c.4219G > A E1407K 1 (1%) 1/1 c.4297_4299delGAG E1433del 1 (1%) N/Ac.4390C > G R1464G 1 (1%) N/A c.4480G > A G1494R 1 (1%) N/A c.4657G > CA1553P 1 (1%) 1/1

It was then sought to define the spectrum and frequency of somatic ALKmutations across all neuroblastoma phenotypic subsets. A representativeset was identified of 594 primary neuroblastomas obtained at diagnosisfor sequence analysis restricted to eight amplicons covering the entiretyrosine kinase domain. In total, non-synonymous sequence variationswere identified in 7.2% of samples (43/594), which were grouped intofour hotspots within the kinase domain (Table 2). All putative mutationsin the ALK tyrosine kinase domain were not present in the singlenucleotide polymorphism database (dbSNP;www.ncbi.nlm.nih.gov/projects/SNP/), and were not detected in directsequencing of the ALK tyrosine kinase domain in 218 normal controlalleles. The most prevalent mutation resulted in an arginine toglutamine substitution at amino acid position 1275 (R1275Q) and occurredin 20/43 tumors with mutations (47%). This was also the most commongermline mutation discovered in hereditary neuroblastoma pedigrees, butwas acquired in all 17 sporadic cases here where matched germline DNAwas available. The second most common mutation resulted in aphenyalanine to leucine substitution at amino acid position 1174 (F1174L), occurring in 7/43 tumors harboring any sequence variation. Inaddition, the 1174 phenylalanine codon was mutated to isoleucine orcysteine in one case each, so that this codon was altered in 21% oftumors with a tyrosine kinase mutation. Of the 43 kinase domainmutations discovered in tumor tissues, constitutional DNA was availablefor 32 patients, and the majority were somatically acquired with 6.3% ofcases (2/32) showing the same alteration in the germline (L1204F and11250T; Table 2).

ALK Amplification and Regional Gain of the ALK Locus are Associated withIncreased ALK ExpressionTo define ALK allelic status, an overlapping set of 591 primaryneuroblastoma tumors was characterized on the Illumina Human Hap550K SNPmicroarray. High-level amplification of ALK was detected in 2.4% oftumors (Table 3), defined as copy number >4.5 of the entire ALK generelative to the chromosome 2 copy-number. Additional tumor DNA wasavailable for 10 of these cases, and none showed an ALK mutationsuggesting that mutation and gene amplification may be mutuallyexclusive genomic events. All tumors with ALK amplification alsoharbored MYCN amplification, but intervening sequence between these twogenes located 13 Mb apart often was not co-amplified (FIG. 10A). Asubset of 96 of these tumors was also assayed using the 11luminaExpression Human6 v2 microarray. ALK amplification was significantlyassociated with ALK overexpression (P<0.0001; FIG. 10B). In addition,regional gain of a 40MB region containing both MYCN and ALK was alsoassociated with a significant, but more modest, increase in ALKexpression compared to both whole chromosome gain (P=0.008) and normalcopy number (P<0.0001; FIG. 10B).

TABLE 3 Frequency of ALK mutation and amplification in diagnosticprimary neuroblastomas. Mutation Status (N = 593) P- AmplificationStatus (N = 591) P- All Patients Mutation + Mutation − value*Amplification + Amplification − value* All Patients 867  43 (7%)  550(93%) 14  577  Age 0.6871 0.6036 <365 days 283 (33%) 15 (35%) 179 (33%) 4 (29%) 169 (29%) >365 days 580 (67%) 28 (65%) 370 (67%) 10 (71%) 405971%) Unknown  4 0 1 0  3 INSS Tumor 0.2541 0.0786 Stage 1 171 (20%)  7(16%) 138 (25%) 0 (0%)  75 (13%) 2 119 (14%)  8 (19%)  79 (14%) 0 (0%)48 (8%) 3 123 (14%)  6 (14%)  80 (15%)  2 (14%)  85 (15%) 4 390 (45%) 20(47%) 220 (40%) 11 (79%) 321 (56%) 4S 56 (7%) 2 (4%) 31 (6%) 1 (7%) 42(8%) Unknown  8 0 2 0  6 MYCN Status 0.2452 <0.0001 Not Amplified 710(83%) 33 (79%) 456 (84%)  2 (14%) 459 (81%) Amplified 143 (17%)  9 (21%) 88 (16%) 12 (86%) 108 (19%) Unknown 14 1 6 0 10 Shimada 0.7606 0.0502Histopathology Favorable 398 (49%) 22 (54%) 254 (49%)  2 (15%) 224 (41%)Unfavorable 414 (51%) 19 (46%) 262 (51%) 11 (85%) 318 (59%) Unknown 55 234  1 35 DNA ploidy 0.6653 0.0127 Hyperdiploid 517 (65%) 28 (68%) 355(66%)  3 (27%) 331 (65%) Diploid 275 (35%) 13 (32%) 181 (34%)  8 (73%)176 (35%) Unknown 75 2 14  3 71 COG Risk 0.5217 0.0009 Group Low 327(38%) 17 (40%) 237 (43%) 0 (0%) 151 (26%) Intermediate 124 (15%)  7(16%)  72 (14%) 0 (0%)  78 (14%) High 406 (47%) 19 (44%) 237 (43%)  14(100%) 342 (60%) Unknown 10 0 4 0  6 *p-values from Fisher's Exact Test.ALK Mutations Occur in all Phenotypic Subsets, but Amplification isRestricted to the most Aggressive CasesNeuroblastoma is a diverse neoplasm and can behave in either a verybenign fashion, or an extremely malignant one, based on a variety ofclinical and biological factors (Maris et al. (2007) Lancet369:2106-2120). Three risk groups are defined, with low-, intermediate-and high-risk cases having cure rates of >97%, >90% and 40-50%,respectively (Maris et al. (2007) Lancet 369:2106-2120). These curerates are achieved with no chemotherapy in the low-risk group, moderatedose intensity chemotherapy in the intermediate-risk patients, andhighly intensive multimodal chemoradiotherapy for the high-riskpatients. Prior published studies had focused on the ALK status in thehigh-risk group of patients, and here it was determined if ALKaberrations occurred in the more benign subset of cases as well. Asshown in Table 3, ALK mutations occurred in all phenotypic subsets,including all disease stages and risk groups. Mutations were seen in thevery benign state 1 low-risk tumors as well as Stage 4S cases, that showspontaneous complete regression without cytotoxic therapy (D'Angio etal. (1971) Lancet 1:1046-1049). In contrast, ALK amplification eventswere restricted to the high-risk group of patients only. Thus, thelikelihood of an ALK aberration at diagnosis in diagnostic high-riskneuroblastic tissues, where novel therapeutic approaches are needed, isapproximately 11.3%.ALK R1275Q, R1192P, G1128A, and F1174L are Gain-of-Function Mutationsthat Induce Differential Constitutive Kinase ActivationTo determine the functional consequences of common germline and somaticmutations, human ALK cDNAs were engineered harboring the three mostcommon germline mutations (R1275Q, R1192P, G1128A), and the F1174Lmutation that was only seen in tumor tissue. Next these cDNAs, as wellas NPM-ALK and wild-type ALK, were stably overexpressed in retinalpigment epithelial cells immortalized with telomerase reversetranscriptase (hTERT-RPE 1) via retroviral infection. hTERT-RPE1 cellswere chosen for these experiments because they are human neuralcrest-derived, as are neuroblastomas, and do not express detectablelevels of ALK by quantitative RT-PCR. FIG. 11 shows that native ALK wasexpressed in all transfectants, but phosphorylation of the tyrosine 1604residue, indicative of kinase activation, was clearly different based onmutation type. Cells over expressing the R1275Q and F1174L mutations,the two most common somatic mutations observed here, showed the mostintense phosphostaining. Cells over expressing the G1128A mutant showedweak phosphostaining, only observable on prolonged exposure similar toforced overexpression of wild-type ALK. Without being bound by theory,this could provide an explanation, at least in part, for the observationthat this germline mutation was unique to a previously reported largemultiplex family that was notable for having multiple unaffectedcarriers, with the lowest tumor penetrance of all neuroblastoma familiesstudied.Cytotoxicity to Pharmacological ALK Inhibition in Vitro is Dependentupon ALK Genomic StatusIt has been shown that mRNA knockdown of ALK in neuroblastoma cell lineswith mutation or amplification is cytotoxic, and thus ALK inhibitionmight offer a tractable therapeutic target. In addition, it has beenshown herein that pharmacologic inhibition of ALK kinase activity had ananti-proliferative effect in ALK mutated cell lines (see also Chen etal. (2008) Nature 455:971-974; George et al. (2008) Nature 455:975-978;Janoueix-Lerosey et al. (2008) Nature 455:967-970). In order totranslate these findings to the clinic as quickly as possible, thesensitivity of neuroblastoma was determined in in vitro and in vivomodels to PF-02341066, an ATP-competitive, orally bioavailable smallmolecule inhibitor of ALK and MET, that has shown safety in early phaseclinical trials (Kwak et al. (2009) J. Clin. Oncol., 27:15s (abstr3509)).

A panel of eighteen human neuroblastoma-derived cell lines, chosen to berepresentative of ALK genomic status in primary tumors, was utilized todetermine the IC₅₀ of PF-02341066 by concentration-response curvesacross a 4-log dose range (1-10,000 nM). Inhibition of substrateadherent growth was screened in a real-time electrical impedancemonitoring system (Yu et al. (2006) Anal. Chem., 78:35-43; Atienza etal. (2006) J. Biomol. Screen 11:634-643). Cell lines harboring an ALKaberration (mutations or amplification) displayed significantly superiorinhibition of growth than cell lines with wild-type ALK status(P=0.0004, FIG. 12). Cell lines harboring the R1275Q mutation or genomicamplification were more sensitive to this compound than those harboringF1174L mutations (P =0.041).

To demonstrate that cytotoxicity with PF-02341066 is mediated throughALK inhibition, it was shown that phospho-ALK correlated with ALKgenomic status (FIG. 13), and that none of seven neuroblastoma celllines studies, selected to be representative ALK genomic status, showedphospho-MET expression. Furthermore, siRNA knockdown of MET in a panelof 4 cell lines representative of ALK status showed no significantgrowth inhibition, as opposed to the significant inhibition seen withsiRNA mediated ALK knockdown. Finally, it was shown that cytotoxicitywas directly correlated with abrogation of phospho-ALK (see below, andFIG. 13).

Cytotoxicity in Vitro Correlates with Abrogation of Phospho-ALK andDifferential Inhibition of Downstream Signaling PathwaysTo correlate the phenotypic effect seen in vitro to degree of inhibitionof constitutive kinase activation and to elucidate potential mechanismsunderlying differential sensitivity to PF-02341066, dose dependentinhibition of phosphoprotein signaling was determined in three celllines, selected to be representative of ALK genomic status observed inpatient samples. The dose that first corresponded with inhibition ofcell growth was correlated to abrogation of ALK tyr1604 phosphorylationin all three-cell lines (FIG. 13). Inhibition of proliferation andabrogation of phospho-ALK occurred in a dose-dependent manner in the33-100 nM range in NB1 (WT amplification) and NB1643 (R1275Q), but didnot occur in SH-SYSY (F1174L) until 1000 nM, and these data were closelycorrelated to the in vitro cytotoxicity assay results. Taken together,these data indicate that the constitutive ALK activation via F 1174Lmutation may be more difficult to inhibit with PF-02341066.

In NB1 cells, abrogation of phospho-ALK occurred in parallel withabrogation of phosphoprotein signaling in pathways known to be importantin lymphoma models of NPM-ALK mediated transformation: pSTAT3, pAKT, andpERK (FIG. 13). By contrast, in NB 1643 cells, although the inhibitionof proliferation at 33 nM correlated to abrogation of phospho-ALK,similar decreases in pSTAT3, pAKT, and pERK did not occur until highermicro molar doses (FIG. 13). Without being bound by theory, theoccurrence of anti-tumor activity against NB 1643 at doses well belowthose where pSTAT3, pAKT, and pERK were abrogated indicates this cellmodel may mediate ALK oncogenicity through alternative signalingpathways.

Pharmacologic inhibition with PF-02341066 shows potent anti-tumoractivity in vivoIt was then determined if pharmacologic ALK inhibition resulted inanti-tumor activity in xenograft models of neuroblastoma. Apharmacologically relevant dose of PF-02341066 at 100 mg/kg/day for 4weeks was tested against serially passaged human neuroblastomaxenografted in the flank of CB 17 immunodeficient mice (Zou et al.(2007) Cancer Res., 67:4408-4417; Christensen et al. (2007) Mol. CancerTher., 6:3314-3322). PF-03241066 caused regression of all NB1643xenografts (R1275Q) within three weeks, and complete regression wassustained over the fourth week of dosing (P<0.0001, FIG. 14A). On targetactivity was confirmed by demonstrating inhibition of ALKphosphorylation in NB 1643 xenograft protein lysates harvested after 2days of administration of PF-02341066 and 4 hours after last oral dose(FIG. 14A). Due to the range of in vitro sensitivity against cell linesharboring F1174L mutations, two such xenografts with differingsensitivity were tested. Treated SHSY5Y xenografts resulted insignificant tumor growth delay (P<0.0001, FIG. 14B). In contrast,treatment of NBSD xenografts showed no statistically significantdifference in tumor volume over time between treatment and control mice(P=0.3, FIG. 14C).

As cell line protein lysates and archival tumor specimens candemonstrate phospho-ALK staining in the absence of genomic ALKaberrations, in vivo activity of PF-02341066 was compared against twoxenografts with diploid copy number and wild type ALK sequence but withdiffering levels of ALK expression and phospho-ALK activation. NB-EBc1xenografts (previously shown to have weak phospho-ALK staining), treatedwith drug demonstrated significant tumor growth delay (p<0.0001, FIG.14D). By contrast, SKNAS xenografts with absent ALK expression and nodetectable phospho-ALK showed no tumor growth delay (P=0.87) and anon-significant difference in days to reach a tumor volume of 1.5 cm³(P=0.7) (FIG. 14E).

Homology Modeling Predicts a Structural Basis for Differential ActivityAgainst R1275Q and F1174L Mutations

A computational homology model of PF-02341066 bound to ALK was derivedfrom the available co-crystal structure of PF-02341066 bound to thekinase domain of c-Met. Modeling was possible due to the high sequencehomology of the kinase domains in the areas near the inhibitor-bindingsite. This model predicts that PF-02341066 binds very similarly to bothALK and c-Met, with nearly all of the major protein inhibitorinteractions being conserved. In the PF-02341066 bound conformation ofwild-type ALK, residues D1270 through Y1278 of the kinase activationloop form a bend. This bend creates one end of the inhibitor-bindingsite and positions the side chain of Y1278 to enable a key bindinginteraction with the fluorodichlorophenyl group of PF-02341066 (FIG. 15A, B).

The ALK homology model predicts that a R1275Q mutant is likely to havethe same PF-02341066 bound activation loop conformation as wild-type ALKand c-Met. The side chain of R1275 points out towards solvent and doesnot appear to make interactions critical for stabilizing the PF-02341066bound activation loop conformation (FIG. 15C). Therefore substitution ofarginine to glutamine is predicted to be accommodated on the proteinsurface, and is not predicted to result in an activation loopconformation that would significantly decrease key binding interactionswith PF-02341066.

In contrast, the F1174L mutation is predicted to result in an activationloop conformation that significantly decreases binding of PF-02341066.The side chain of F1174 is situated in a cluster of three phenylalanines(F1174, F 1245, and F 1271) in attractive van der Waals contact witheach other. The three phenylalanines appear to form an aromatic centerthat is part of a larger hydrophobic core. This hydrophobic core islikely important for stabilizing the particular activation loopconformation necessary to make key binding interactions with PF-02341066(FIG. 15 A, D). One of the three phenylalanines in the hydrophobic core,F1271, is at the beginning of the activation loop and lies within aconserved DFG (Aspartic acid, Phenylalanine, Glycine) amino acidsequence. Conformational flipping of this conserved DFG sequences isknown to effect large changes in activation loop conformations in manytyrosine kinases (Lu et al. (2009) Biochemistry 48:3600-3609; Hubbard,S.R. (2002) Front. Biosci., 7:d330-340; Han et al. (2009) J. Biol.Chem., 284:13193-13201).

As shown in FIG. 15D, a F1174L mutation is predicted to result in adecrease in interactions within the aromatic center. This occurs becausethe smaller size and non-planarity of leucine compared to phenylalanineis predicted to result in a loss of contact with F1245. The loss of theF1174L-F1245 interaction and accommodation of the non-planar leucinelikely destabilizes the aromatic center and hydrophobic core formed bythe three phenylalanines in wild type ALK. Destabilization of thehydrophobic core could result in a flip of a flexible amino acidsegment, D1270-F1271-G1272, resulting in a large positional change inthe activation loop such that key binding interactions to PF-02341066are lost and inhibitory activity is reduced.

The recent discovery of germline and somatic gain of function mutationsin the receptor tyrosine kinase ALK provides a tractable therapeutictarget for new drug development in neuroblastoma. Here, the frequencyand spectrum of ALK gene mutations and amplification events wasdetermined in a representative series of diagnostic primaryneuroblastomas. The instant results demonstrate that ALK mutations aredetected in 7% of cases and are found primarily in the tyrosine kinasedomain. Other kinases such as EGFR (Lee et al. (2006) PLoS Med 3:e485),KIT (Gari et al. (1999) Br. J. Haematol., 105:894-900), and FLT3(Frohling et al. (2007) Cancer Cell 12:501-513) show activatingmutations in both the intra- and extra-cellular and juxta-membranedomains. Accordingly, it is clear that sequence alteration outside thekinase domain may be functionally relevant, but it is clear from thedata presented herein that the majority of ALK mutations occur in thekinase domain.

High-level amplification of ALK was detected in another 2.4% of tumors,so that about 10% of neuroblastomas have genomic evidence for ALKactivation at diagnosis. Unlike sequence variation mutations, high-levelALK amplification was restricted to the high-risk group of tumors withconcomitant MYCN amplification.

These data indicate that in the setting of genomic instability, ALK canbe a target for amplification events that presumably lead to pathwayactivation due to homodimerization of overexpressed ALK protein.Notably, 19% of tumors show unbalanced gain of 2p including the ALKlocus. Some of these are focal, suggesting a tandem duplication eventtargeting the ALK locus, but the majority of these are very large, ofteninvolving the majority of the short arm. The unbalanced gains in 2p maycorrelate with pathway activation and/or sensitivity to targetedinterruption of ALK signaling.

The two most commonly observed mutations resulted in robust constitutivephosphorylation of the ALK kinase, whereas the rare germline mutationG1128A, which has not been observed somatically, resulted in only weakactivation, similar to the amount seen with forced overexpression ofwild-type protein. While these data are semiquantitative, the magnitudeof difference is clear and consistent with the observation thatheritable G1128A mutations resulted in low tumor penetrance, compared tothe families with R1275Q germline mutations, each showing near completetumor penetrance in at risk carriers. The hTERT-rRPE 1 assay providescomplimentary information to forced overexpression in other systems,such as BAF3 (George et al. (2008) Nature 455:975-978) or NIH-3T3 cells(Janoueix-Lerosey et al. (2008) Nature 455:967-970), but the instantsystem offers advantages for further functional analysis of mutations asthey are discovered to determine their potential as an oncogenic driverin an appropriate cellular context. Cellular context is important andthe use of this system to understand the functional consequences of allsequence variations identified may be used to dissect the oncogenicpotency of the various mutations discovered.

The data clearly demonstrate that cytotoxicity to PF-02341066 is highlyassociated with ALK genomic status and evidence for constitutiveactivation. It is also evident that some neuroblastomas may somaticallyactivate ALK signaling in the absence of mutation or amplification.

PF-02341066 has already demonstrated safety and tolerability in humans,as well as dramatic reductions in tumor volumes and diseasestabilization for non-small cell lung cancers with activated ALK viatranslocation events. This drug is robustly cytotoxic in vitro and invivo in cells with the most common mutation (R1275Q) and in wild-typecells with high-level ALK amplification (p <0.0004), and it is shownherein that this is not an effect of c-Met inhibition. Fl 174L modelsalso show growth inhibition with PF-02341066, though not nearly aspotently; and there are models without evidence for ALK mutation, butwith constitutive activation that show growth inhibition, suggestingthat several subsets of patients may benefit from ALK inhibitiontherapy. It is evident that phospho-ALK is an appropriate surrogatebiomarker of response and that abrogation of phospho-ALK can be highlycorrelated with response to pharmacologic inhibition.

In NPM-ALK driven lymphoma models, it has been shown that severalcanonical signaling pathways are activated, including STAT3, AKT/PI3K,and RAS/ERK, thus influencing cell proliferation and survival (Zamo etal. (2002) Oncogene 21:1038-1047; Chiarle et al. (2005) Nat. Med.,11:623-629; Lim et al.

(2009) Blood 114:1585-1595). However, the situation appears to be morecomplex in neuroblastoma. The NB1 model with wild type, amplified ALK,shows constitutive activation of each of these pathways, withdose-dependant abrogation of signaling paralleling diminution ofphosphorylated ALK and cytotoxicity. However, the NB1643 cells harboringan R1275Q mutation are equally sensitive to PF-02341066, show the samepattern of diminution of phospho-ALK staining, but do not likewiseabolish phosphorylated proteins in the STAT3, AKT/PI3K, and RAS/ERKuntil much higher doses. SY5Y cells, which are relatively resistant toPF-02341066, also do not abolish STAT3, AKT/PI3K, and RAS/ERK signalingeven at the higher dose levels where cytotoxicity (and diminishedphosphorylated ALK) was seen. Without being bound by theory, thesedata—taken together—indicate that ALK mutations may exert theironcogenic effect through other pathways.

The data presented herein show that mutations in ALK are present acrossall neuroblastoma disease subsets, both benign and malignant forms,consistent with acquired ALK activation being an early event intumorigenesis. ALK amplification, however, is strongly associated withthe high-risk subset (P<0.001) and MYCN amplification (P<0.001), so thatapproximately 11% of all newly diagnosed high-risk neuroblastomapatients harbor genetic evidence for ALK activation and can be expectedto potentially benefit from ALK inhibition therapy. In the instantdataset, mutations and amplifications of ALK are mutually exclusive,suggesting these modes of genomic dysregulation do not co-occur insporadic neuroblastoma. Mutations in ALK are significantly more frequentin human neuroblastoma-derived cell lines. Without being bound bytheory, this may occur through selection of rare clones that are presentin diagnostic tissues and emerge during therapy, as has been shown inchronic myeloid leukemias harboring a BCR-ABL gene translocation,mutation or amplification (Gorre et al. (2001) Science 293:876-880).

As the crystal structure for the ALK kinase domain has not been solved,in silico techniques were used to explore the observed differentiallycytotoxicity of PF-02341066 against the two most common mutationsobserved in patient samples.

Structural modeling predicted that the phenylalanine to leucinesubstitution at codon 1174 (F1174L) results in destabilization of thePF-02341066 binding site, whereas the R1275Q mutation has no predictedeffect on this small molecule binding and thus competing with ATP.

Taken together, these data provide strong rationale for the clinical useof PF-02341066 for patients with neuroblastoma. This represents thefirst therapy for neuroblastoma specifically developed for a mutatedoncogenic driver.

EXAMPLE 4 Table 4 provides the frequency and spectrum of ALK mutation indiagnostic primary neuroblastomas (n=1148), as described hereinabove.Table 5 provides the frequency of mutations in various subsets of thepopulation based on risk, as described hereinabove.

TABLE 4 Frequency and spectrum of ALK mutations in diagnostic primaryneuroblastomas. All Patients Mutation+ All Patients 1148 84 (7.3%) Age<365 days 438 (38%) 29 (6.6%) >365 days 709 (62%) 55 (7.8%) >3650 days36 (3%) 6 (17%) INSS Tumor Stage 1 288 (25%) 14 (4.9%) 2 211 (18%) 21(10%) 3 172 (15%) 10 (5.8%) 4 401 (35%) 36 (9%) 4S 76 (7%) 3 (4%) MYCNStatus Not Amplified 984 (86%) 65 (6.6%) Amplified 156 (14%) 18 (11.5%)Shimada Histopathology Favorable 629 (55%) 43 (6.8%) Unfavorable 466(41%) 37 (7.9%) DNA Ploidy Diploid 377 (33%) 31 (8.2%) Hyperdiploid 729(64%) 51 (7%) COG Risk Group Low 548 (48%) 35 (6.4%) Intermediate 203(18%) 14 (6.9%) High 397 (34%) 35 (8.8%)

TABLE 5 Frequency of mutations in low risk, intermediate risk, and highrisk samples. SAMPLE TOTAL SET MUTATIONS R1275 F1174 F1245 LOW RISK35/548 (6.4%) 16/36 (44%) 7/23 (30%) 6/11 (55%) (N = 548) INT RISK14/203 (6.9%) 6/36 (17%) 6/23 (26%) 1/11 (10%) (N = 203) HIGH RISK35/397 (8.8%) 14/36 (39%) 10/23 (43%) 4/11 (36%) (N = 397) ALL 84/1148(7.3%) 36/84 (43%) 23/84 (27%) 11/84 (13%) TUMORS (N = 1148)

EXAMPLE 5

The co-administration of ALK antibodies with a tyrosine kinase inhibitorinduces cell death in cells that were less sensitive to the tyrosinekinase inhibitor alone. FIG. 16 shows the co-administration of PF-1066with mAb 30 and 49 were significantly more effective than PF-1066 aloneor mAb 30 and 49 alone against SH-Sy5Y (F1174L) cells. mAb 30 and 49 aredescribed in Moog-Lutz et al. (J. Biol. Chem., (2005) 280:26039-26048).

EXAMPLE 6

Anaplastic Lymphoma Kinase (ALK) was originally identified in anoncogenic fusion protein expressed in anaplastic large cell lymphoma(ALCL) as a result of a chromosomal translocation (Morris et al. (1994)Science 263:1281-4). This fusion links the intracellular region of theALK receptor tyrosine kinase (RTK) to the N-terminal portion ofnucleophosmin (NPM), leading to constitutive kinase activation in anNPM-ALK fusion (Morris et al. (1994) Science 263:1281-4). Otheroncogenic ALK fusions have since been identified in several humancancers, including non-small cell lung cancer (NSCLC), squamous cellcarcinoma, and inflammatory myofibroblastic tumors (Soda et al. (2007)Nature 448:561-6; Rikova et al. (2007) Cell 131:1190-203; Jazii et al.(2006) World J. Gastroenterol., 12:7104-12; Griffin et al. (1999) CancerRes., 59:2776-80). The full-length ALK RTK has also been linked toneuroblastoma, a pediatric cancer of the sympathetic nervous system thataccounts for 10% of childhood cancer mortality (Miyake et al. (2002)Oncogene 21:5823-34; Maris et al. (2010) N. Engl. J. Med., 362:2202-11;Smith et al. (2010) J. Clin. Oncol., 28:2625-34). Intact ALK isexpressed in the majority of neuroblastomas, suggesting a role inautonomic nerve development, and the ALK gene is amplified in 2-3% ofcases (Lamant et al. (2000) Am. J. Pathol., 156:171 1-21; Passoni et al.(2009) Cancer Res., 69:7338-46; Wang et al. (2006) Cancer Res.,66,6050-62; George et al. (2007) PLoS One 2:e255). Herein, activatingmutations within the tyrosine kinase domain of full-length ALK wereidentified as the major cause of familial neuroblastoma and were alsoshown to arise somatically in up to 10% of sporadic neuroblastoma cases(see also Janoueix-Lerosey et al. (2008) Nature 455:967-70; Mosse et al.(2008) Nature 455:930-5). Amplification and mutation of ALK lead to itsconstitutive autophosphorylation and activation, and may be associatedwith a more aggressive clinical course (see also Passoni et al. (2009)Cancer Res., 69:7338-46; George et al.

(2007) PLoS One 2:e255; Mosse et al. (2008) Nature 455:930-5;Osajima-Hakomori et al. (2005) Am. J. Pathol., 167:213-22; De Brouwer etal. (2010) Clin. Cancer Res., 16:4353-62).

Cure rates among children with high-risk neuroblastoma have shown onlymodest improvement, despite dramatic escalations in the intensity oftherapy provided. Survivors of modern high-risk neuroblastoma therapyare at risk for major morbidities, many of which can be life-threatening(Hobbie et al. (2008) Pediatr. Blood Cancer 51:679-83; Oeffinger et al.(2006) N. Engl. J. Med., 355:1572-82). It is therefore clear that newapproaches are required for treating neuroblastoma.

Materials and Methods Cell Culture and Reagents

All cell lines were maintained in a 5% CO₂ incubator at 37° C. in RPMI(Invitrogen) supplemented with 10% fetal calf serum, L-glutamine,penicillin/streptomycin, and gentamicin (Invitrogen). Mouse monoclonalIgG1 antibodies 14, 30, 46 and 49 were generated to the extracellulardomain of human ALK as previously described (Moog-Lutz et al. (2005) J.Biol. Chem., 280:26039-48). Crizotinib was obtained from Pfizer anddiluted in dimethylsulfoxide (DMSO; Sigma) for use in cell culture.Murine IgG1 (Sigma-Aldrich) and DMSO were used respectively as negativecontrols.

Quantitative mRNA Expression

RNA from primary diagnostic tumor specimens of 229 children withneuroblastoma was obtained and analyzed using Affymetrix Human ExonArray (HuEx). HuEx arrays were normalized using quantile normalizationand summarized using robust multichip average (RMA) using AffymetrixPower Tools software package version 1.12. ALK expression levels wereobtained by averaging the core unique probesets for the ALK transcript(Transcript ID: 2546409). Data were analyzed for significance amongpatients diagnosed with low risk disease (stage 1 and 2 neuroblastoma, n=24), high risk metastatic MYCN non-amplified neuroblastoma (>18 monthsof age at diagnosis, n =141), and high risk MYCN amplified neuroblastoma(n=64). The overall p-value among the groups was carried out usinglikelihood ratio chi-squared tests in the context of a general linearmodel. Differences between two subgroups were assessed using the Waldtest. The analyses were performed using the R statistical package (Rversion 12.1).

Immunohisiochemistry

Cases were selected from a review of neuroblastic tumors accessioned tothe Pathology Department of the Children's Hospital of Philadelphia from1987 to 2004. Selection of specimens and construction of the tissuemicro-array (TMA) followed approval by the Children's Hospital ofPhiladelphia Institutional Review Board. All tumors were reviewed by apediatric pathologist for adequacy, and classified pathologically fordiagnosis, grade, Shimada histology and mitotic-karyorrhectic indexusing International Neuroblastoma Pathology Classification criteria. Theresultant TMA comprised two paraffin blocks and contained tumor coresfrom 126 patients, including 117 neuroblastomas and 9 nodularganglioneuroblastomas. 0.6 mm cores of representative tumor tissue fromeach case were used to construct the tissue microarray blocks using amanual arrayer (Beecher Instruments). MYCN amplification status andstage (International Neuroblastoma Staging System, INSS) were obtainedby tumor registry review. Staining of the TMA was conducted withprediluted anti-ALK-1 (Ventana Medical Systems), using pressure cookerantigen retrieval, overnight incubation, and avidin-biotin complexconjugation. Percent positivity of the neuroblasts within the cores wasassessed, and intensity of staining was graded on a scale of 1-3 (weak,moderate, strong). An ALK staining score was calculated as the productof percentage of neuroblasts stained and grade of intensity.

Immunofluorescence

Cell lines were plated on Lab Tek II Chamber Slides (Thermo Scientific).

After overnight incubation at 37° C., slides were transferred to ice andcells were washed three times with ice-cold PBS before blocking for 20minutes with 10% BSA in PBS. Cells were next incubated for 60 minutes ona rocker table with ALK antibody mAb14 at a 1:100 dilution in 1.5%BSA/PBS. After three 5 minutes washes, cells were incubated for 45minutes in the dark with Rhodamine-conjugated goat anti-mouse secondaryantibody (Jackson) then washed 3×5 minutes and coverslipped withmounting media containing DAPI stain (Santa Cruz) for evaluation with afluorescence microscope.

Flow Cytometry with ALK Antibodies

Cells were kept ice-cold during staining to minimize receptorendocytosis. Cells that had achieved 70-80% confluency were harvestedand washed twice with ice-cold 1% FCS/PBS buffer containing 2 mM EDTA(Invitrogen), then ALK antibody mAb14 was added as a staining antibodyat a final concentration of 10 μg/ml. Cells were incubated 20 minutes onice then washed twice. Donkey anti-mouse IgG PE (eBioscience) was thenadded at a concentration of 2.5 μg/ml and cells were incubated for 20minutes on ice in the dark and washed. In some experiments, cells weregrown in T25 flasks until 70-80% confluency and crizotinib was added (orDMSO as a negative control) and cells harvested at various subsequenttimepoints for flow cytometry. In some experiments, staining wasconducted with both the mAb14 and mAb46 which binds a distinct epitopefrom mAb14. Cells were then analyzed on an LSR II Flow Cytometer (BDBiosciences). All results shown are representative of at least 3independent experiments.

Growth Inhibition

The Real-Time Cell Electronic Sensing (RT-CES) system (ACEA Biosciences,San Diego, Calif.) was used to measure the in vitro effect of the ALKantibodies mAb30 and mAb49 alone or in combination with the tyrosinekinase inhibitor crizotinib on neuroblastoma cell line growth. Celllines were plated in triplicate in 96-well plates. Antibody and/or drugwere first added 24 hours after plating, and antibody treatment wascontinued for four additional days. Growth inhibition was calculated as:100*(1−(cell index treatment/cell index control)). All cell lines wereroutinely mycoplasma tested and genotyped using the AmpFLSTR Identifilerkit (Applied Biosystems). All RT-CES experiments were conducted aminimum of three times, and results quoted as mean ± standard deviation.For statistical analysis of results, linear mixed effect models werefitted to examine time and treatment effects. To account fornonlinearity, time by treatment and time square by treatment interactionterms were included in the models. F tests were used to examine thedifference of the progression of cell index over time between eachindividual treatment and the combination treatment.

Calculation of IC₅₀

The RT-CES system was used to measure growth of cell lines plated in a5-log dose range of 1-10,000 nM crizotinib alone or in combination with10 μg/ml ALK mAb30 and mAb49. GraphPad Prism Version 5.0 was used tocalculate the IC₅₀ from the RT-CES-generated cell index data using thelog(inhibitor) vs. response—variable slope equation.

Western Blots

Cells were grown in 10 cm dishes until 70-80% confluency, at which point1000 nM crizotinib, 10 μg/ml ALK antibody, both agents, or the relevantnegative controls were added. After 72 hours of incubation, flasks weretransferred to ice, washed with ice cold PBS, and cells scraped intoice-cold PBS. After pelleting, whole cell lysates were harvested andimmunoblotted with either antibodies against ALK (1:1,000; CellSignaling, 3333), phospho-ALK Tyr 1604 (1:1,000, Cell Signaling, 3341),or actin (1:2000; Santa Cruz, sc-1616). Results shown are representativeof two independent experiments.

Propidium Iodide Staining

SY5Y cells were plated in 6-well plates at 2×105 cells per well. After24 hours, 1000 nM crizotinib, 10 μg/ml ALK antibody, both agents, or theappropriate negative controls, were added. Antibody treatment wascontinued for four additional days. Cells were then harvested and washedtwice. Ice-cold ethanol was added while vortexing, and cells wereallowed to stand for 20 minutes at 4° C. Cells were again washed twiceand then resuspended in phosphate-citric acid buffer and allowed tostand at room temperature for 5 minutes. After washing, cells wereresuspended in a 1% FBS solution containing RNAseA (Roche) and 50 μg/mlpropidium iodide (Sigma-Aldrich) and incubated at room temperature inthe dark for 30 minutes. Cells were then analyzed on an LSR II flowcytometer.

Antibody-Dependent Cell-Mediated Cytotoxicity Assay

The Cyto Tox 96 Non-Radioactive Cytotoxicity Assay (Promega) was used toassess antibody-dependent cell mediated cytotoxicity. Normal donorperipheral blood mononuclear cells were used as effectors and plated in10 cm dishes in complete RPMI for 2 hours at 37° C. to allow monocytesto adhere. Non-adherent peripheral blood lymphocytes were then replatedin complete RPMI containing 1000 IU/ml rhIL-2 (Chiron) and incubatedovernight. The following day, effector cells were collected and washed.Target NB1 or SY5Y cells that had been grown on T75 flasks until 70-80%confluency were also harvested and washed. In some experiments, SY5Ycells were pre-incubated for 48 hours in crizotinib before being used astarget cells in the ADCC. Effector and target cells were then plated inquadruplicate at effector:target ratios of 50:1, 25:1, and 10:1 andexperimental wells treated with 1 μg/ml ALK mAb30 and mAb49. Controlwells were plated according to the manufacturer's specifications. Aftera 4-hour incubation at 37° C., cell viability was assessed by anenzymatic assay allowing for the quantification of lactate dehydrogenasewhich is released upon cell lysis. The % cytotoxicity was calculated asfollows: [(Experimental−Effector Spontaneous−Target Spontaneous)/(TargetMaximum−Target Spontaneous)]×100. To ensure specificity of the ALKantibody, separate experiments were conducted comparing untreated wellsto treatment with 1 μg/ml murine IgG1. No difference was detectedbetween untreated (mean % cytotoxicity=3.1%, SD=0.3%) and IgG1-treatedwells (mean % cytotoxicity=0.64%, SD=0.28%; p=0.8822). Results shown arerepresentative of three independent experiments, and quoted as mean±standard deviation.

Results ALK is Widely Expressed in Neuroblastoma Tumors and Cell Lines

Successful immunotherapy requires the targeted antigen to be expressedselectively in tumors, but not in normal tissue. Moreover, the targetedantigen must be expressed on the majority of tumors for immunotherapy tobe relevant to a large proportion of patients, and expression levelsshould ideally be related directly to measures of disease severity.Since intact ALK is normally found only in the developing embryonic andneonatal brain (Iwahara et al. (1997) Oncogene 14:439-49), it could be avaluable target for immunotherapy if expressed in the majority oftumors. To assess ALK expression in primary patient tumors, data fromWang et al. (Cancer Res. (2006) 66:6050-62) as well as the TARGETinitiative (Therapeutically Applicable Research to Generate EffectiveTreatments: target.cancer.gov/). As shown in FIG. 17A, ALK mRNAexpression is seen in tumors from patients with clinically aggressivedisease, especially in those with high-risk metastatic disease and/orMYCN amplification [p=5.06E-05 for high-risk MYCN amplified (HRA) vs.low-risk (LR), p=0.0022 for HRA vs. high-risk MYCN non-amplified (HRN),p=0.0211 for HRN vs. LR]. To verify ALK expression at the protein level,a tissue microarray of diagnostic neuroblastomas andganglio-neuroblastomas was analyzed. Samples from 126 patients werestained for native ALK expression with anti-ALK-1 and graded theintensity of ALK staining on a scale of 1-3 (representative samplesshown in FIG. 17B) and percent positivity. Among the samples analyzed,109 (86.5%) were ALK-positive, with 75 samples (59.5%) having eithermoderate (grade 2) or strong (grade 3) staining. Moreover, as shown inFIG. 17C, ALK expression was significantly stronger in patients withINSS stage 4 (p=0.0108; top panel) or amplified MYCN (p=0.0065; bottompanel). These data indicate that ALK is expressed in the vast majorityof neuroblastoma tumors, and that expression levels are higher in thosepatients with the worst prognosis.

Since ALK expression at the cell surface is a prerequisite forALK-targeted immunotherapy to be effective, flow cytometry was used toquantify cell surface ALK levels in a panel of humanneuroblastoma-derived cell lines. As shown in FIGS. 17D and 17E, ALK wasabundant on the cell surface of cell lines expressing either wild-typeALK (NB1, EBc1, and IMR5) or mutated ALK (1643, R1275Q; SY5Y, F1174L;Kelly, F1174L) when compared to the negative control SKNAS line that hasbeen shown to have no detectable ALK expression (see above and Mosse etal. (2008) Nature 455:930-5). As also shown in FIG. 17E, cell surfaceALK expression corresponded closely with relative ALK mRNA expression asmeasured by quantitative RT-PCR. To further analyze cell surface ALKexpression, immunofluorescence staining of the neuroblastoma cell linesNB1 and SY5Y was conducted (FIG. 17F). Consistent with the results offlow cytometry, ALK was detected at the plasma membrane of both celllines. These data indicate widespread expression and cell surfacelocalization of full-length ALK in neuroblastoma.

An ALK Antibody Induces Growth Inhibition and Cytotoxicity

Having established that extracellular ALK antigens are accessible at thesurface of neuroblastoma cell lines, it was then determined whether anantagonistic ALK monoclonal antibody identified by Moog-Lutz et al. (J.Biol. Chem. (2005) 280:26039-48) can inhibit growth of a neuroblastomacell line driven by expression of activated ALK. First SY5Y cells (whichexpress F1174L-mutated ALK) were treated with a 3-log dose range of amixture of two ALK antibodies (mAb30 and mAb49), and growth was measuredover 152 hours relative to control-treated cells (FIG. 18A). AlthoughSY5Y cells express high levels of phospho-ALK, they are relativelyinsensitive to growth inhibition by crizotinib. However, treatment withthe ALK antibody resulted in significant dose-dependent growthinhibition as compared to cells treated with control immunoglobulin. Toassess whether this effect is general, a panel of well-characterizedneuroblastoma cell line models harboring wild-type ALK (IMR5), mutatedALK (1643 and SY5Y), amplified ALK (NB1), or undetectable ALK (SKNAS)were treated with a fixed concentration of mAb30 and mAb49 (10 μg/mltotal). As shown in FIG. 18B, there was a direct correlation between ALKexpression levels and antibody-induced cytotoxicity, with the ALKamplified line NB1 showing the greatest sensitivity to antibodytreatment, and the ALK-negative cell line SKNAS showing no growthinhibition. As an additional negative control, retinal pigmentedepithelial cells (RPE-1)—a non-neuroblastoma, ALK-negative, neuralcrest-derived cell line—was treated with 10 μg/ml ALK antibody or murineimmunoglobulin as above, and saw no growth inhibition (FIG. 18C).Importantly, given their variable sensitivity to ALK-targeted TKIs, celllines expressing mutated ALK (1643 and SY5Y) showed significant degreesof antibody-induced cytotoxicity.

Immune cell-mediated ADCC has been shown to be important for themechanism of action of the GD2 antibody in neuroblastoma, and thiseffect is substantially enhanced in the presence of interleukin-2 (IL-2)(Hank et al. (1990) Cancer Res., 50:5234-9). To explore whether an ALKantibody is also capable of inducing an immune-mediated anti-tumorresponse in neuroblastoma, in vitro ADCC assays were conducted in whichnormal donor peripheral blood lymphocytes (PBL) were used as effectors,and neuroblastoma cell lines used as targets. Lymphocytes pre-incubatedwith IL-2 induced substantially higher levels of cytotoxicity in NB 1cells treated with anti-ALK mAb30 and mAb49 than in untreated cells(FIG. 18D, left panel). F1174L-expressing SY5Y cells also showedcytotoxicity in this assay (FIG. 18D, middle panel), although less thanseen for NB1 cells, possibly because of the lower cell surface ALKlevels seen by flow cytometry (FIG. 17E). No ADCC was detected whenSKNAS cells (untreated or antibody-treated) were used as targets (FIG.18D, right panel), consistent with their lack of ALK expression. Thus,targeting ALK with an antagonistic antibody can effectively inhibitgrowth and induce cytotoxicity of neuroblastoma cell lines harboringeither wild-type or mutated forms of activated ALK.

Crizotinib Treatment Induces Cell Surface Accumulation of ALK

Treatment with a combination of TKIs and therapeutic antibodies thatboth target the same tumor antigen, such as EGFR (Regales et al. (2009)J. Clin. Invest., 119:3000-10) or HER2 (Scaltriti et al. (2009) Oncogene28:803-14), has been shown to enhance the degree of tumor growthinhibition and cytotoxicity that can be elicited by either agent alone.One possible mechanism for this is increased cell surface accumulationof the targeted RTK that results from RTK stabilization upon TKI binding(Bougherara et al. (2009) Mol. Cancer Res., 7:1525-33; Tabone-Eglingeret al. (2008) Clin. Cancer Res., 14:2285-94). Certain activating RTKmutations, including several in c-Kit, may destabilize the RTK and causeintracellular (likely endoplasmic reticulum) accumulation that can bereversed by TKI treatment—leading to increased cell surface expression(Bougherara et al. (2009) Mol. Cancer Res., 7:1525-33; Tabone-Eglingeret al. (2008) Clin. Cancer Res., 14:2285-94). The relatively reducedsensitivity of F1174L ALK expressing SY5Y cells to ALK antibodytreatment (FIG. 18B, 18D) prompted us to investigate a similarpossibility for ALK.

To explore the effects of TKIs on surface ALK levels in neuroblastomacells, SY5Y cells were treated with a range of crizotinib doses (orvehicle). After 72 hours, cells were harvested and analyzed for cellsurface ALK expression by flow cytometry. As shown in FIG. 19A,treatment with 1000 nM crizotinib resulted in a substantial increase incell surface ALK levels for SY5Y cells, with a 51.4% increase in meanfluorescence intensity for cells treated with 1000 nM crizotinib(MFI=636) compared with vehicle treated SY5Y (MFI=420), or isotypecontrol-stained (MFI=90). The enhancement in cell surface ALK levels wasdose dependent (FIG. 19B), and increased with the time of crizotinibexposure (FIG. 19C). Up-regulation of cell surface ALK levels incrizotinib-treated cells was first evident at approximately 8 hourspost-treatment, and continued to increase over the 72 hour periodtested. The possibility that crizotinib binding might induceconformational changes in ALK that could stabilize or expose the epitopeto which the staining antibody was binding—increasing antibody bindingand thus the fluorescence signal in flow cytometry—was also considered.To control for this possibility, the experiment was repeated using asecond anti-ALK antibody (mAb46) known to bind to an epitope distantfrom that of mAb14 (Moog-Lutz et al. (2005) J. Biol. Chem.,280:26039-48). Crizotinib-induced enhancement of cell surface ALK levelswas also observed in this experiment (FIG. 19D), indicating thatcrizotinib promotes cell surface accumulation of its target RTK.

Crizotinib Sensitizes Cells to Growth Inhibition by ALK AntibodyTreatment

To test the hypothesis that crizotinib-induced accumulation of cellsurface ALK sensitizes cells to ALK antibody treatment, the ability ofthe antagonist ALK antibody to inhibit growth of SY5Y cells alone or incombination with crizotinib was compared. As shown in FIG. 20A,treatment with either crizotinib alone (at a sub-IC₅₀ dose of 333 nM) orALK antibody alone (at 10 μg/ml) led to measurable growth inhibition.However, combined treatment with both TKI and ALK antibody had asignificantly larger inhibitory effect as compared to TKI alone(p<0.0001) or antibody alone (p<0.001), leading to almost completegrowth inhibition of SY5Y cells in vitro. Increases in total ALK levelswere also seen by Western blotting (FIG. 20B) in cells treated withcrizotinib (alone or in combination with ALK antibody), consistent withthe flow cytometry results shown in FIG. 19. On the other hand, levelsof phosphorylated ALK were substantially diminished by treatment withcrizotinib, either alone or together with antibody (FIG. 20B),suggesting that the TKI stabilizes ALK while simultaneously blocking itsactivation. It also considered whether crizotinib-induced up-regulationof cell surface ALK might promote ADCC-mediated effects of the ALKantibody. To address this, the in vitro ADCC assays was repeated usinglymphocytes as effectors and SY5Y cells pre-incubated with crizotinib orvehicle as targets. As shown in FIG. 20C, crizotinib pre-incubationsignificantly increased ADCC at effector to target ratios of 50:1(crizotinib-treated mean=55.7±9.3%; vehicle-treated mean=31.1±14.7%;p=0.0331) and 25:1 (crizotinib-treated mean=33.7±2.5%; vehicle-treatedmean=17.3%±7.9%; p=0.0262), providing further evidence for the abilityof crizotinib to sensitize ALK-mutated neuroblastoma cells to ALKantibody treatment.

ALK Antibody Improves Sensitivity to a Broad Range of Crizotinib Doses

Since the previous experiments were conducted at a fixed dose ofcrizotinib, it was then explored whether ALK antibody treatment couldimprove sensitivity of neuroblastoma cells to crizotinib treatmentacross a range of doses. SY5Y cellswere treated with a 4-log range ofcrizotinib doses in the presence or absence of ALK antibody. As shown inFIG. 21A, the addition of ALK antibody significantly enhanced growthinhibition at all crizotinib doses except for the highest dose of 10,000nM, a supra-lethal dose at which the majority of TKI effect is likely tobe off-target. Moreover, these data reveal that combining the ALKantibody shifts the dose-response curve for crizotinib so that a lowdose (10 nM) of crizotinib in combination with anti-ALK induces moregrowth inhibition (mean=33.7±2.8%) than seen with 333 nM crizotinibalone (mean=25.7%, SD=6.5%). As suggested by this finding, antibodytreatment reduced the IC₅₀ for crizotinib treatment of SY5Y cells from3018 nM (crizotinib alone) to 1745 nM for dual therapy (FIG. 21B).

Dual ALK Targeting Enhances Programmed Cell Death

To determine the effect of crizotinib and ALK antibody treatment on cellcycle progression, the impact of TKI and/or antibody exposure on SY5Ycellular DNA content was analyzed by flow cytometry. As shown in FIG.22A, and quantified in FIG. 22B, treatment with antibody alone led to asmall, but significant, increase in the G0/G1 fraction (antibody—treatedmean=69.2±0.5%; vehicle-treated mean=65.7±0.3%) and a small butsignificant decrease in the sub G0 fraction (antibody treatedmean=11.4±0.3%; vehicle-treated mean=13.7±0.2%), suggesting that themain mechanism of antibody action may be G1 arrest. On the other hand,dual antibody and TKI ALK targeting led to large and significantincreases in the sub GO fraction (mAb+crizotinib−treatedmean=38.0±0.4%), suggesting induction of programmed cell death as thedominant mechanism of dual ALK targeting. Treatment with crizotinibalone also increased the sub GO fraction, but to a smaller extent thanseen with dual treatment, consistent with the findings above thatanti-ALK potentiates crizotinib effects.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A method for inhibiting neuroblastoma in a subject comprising theadministration of at least one composition comprising at least one ALKantibody and at least one pharmaceutically acceptable carrier.
 2. Themethod of claim 1, wherein said ALK antibody is immunologically specificfor the extracellular domain of ALK.
 3. The method of claim 1, whereinsaid neuroblastoma is resistant to at least one ALK inhibitor.
 4. Themethod of claim 1, wherein said neuroblastoma is resistant tocrizotinib.
 5. The method of claim 1, further comprising theadministration of at least one composition comprising at least one ALKinhibitor and at least one pharmaceutically acceptable carrier.
 6. Themethod of claim 5, wherein said ALK inhibitor is a small moleculeinhibitor.
 7. The method of claim 6, wherein said ALK inhibitor iscrizotinib.
 8. A kit comprising a first composition comprising at leastone ALK antibody and at least one pharmaceutically acceptable carrierand a second composition comprising at least one ALK inhibitor and atleast one pharmaceutically acceptable carrier.